Thermal reaction device and method for using the same

ABSTRACT

Devices and methods for performing the relative concentration of a target in a sample, the sample containing both target and non-target components, the method performed by partitioning the sample into a large number of reaction volumes such that the target is concentrated relative to the non-target, and performing a detection assay upon each reaction volume to detect the target.

PRIORITY AND CROSS-REFERENCE TO RELATED APPLICATIONS

This international application claims the benefit of U.S. provisionalapplication 60/749,963 filed 12 Dec. 2005, entitled “Devices and Methodsfor Highly Sensitive and Specific Detection of Mutant Alleles,” andfurther claims benefit from U.S. application Ser. No. 11/084,357 filed18 Mar. 2005, which itself is a continuation-in-part application of U.S.application Ser. No. 10/876,046 filed 23 Jun. 2004, which itself is acontinuation-in-part of U.S. application Ser. No. 10/837,885, filed 2May 2004 which itself is a continuation-in-part of 10/818,642, filed 5Apr. 2004, which claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application 60/460,634, filed on Apr. 3, 2003. All ofthe above-referenced applications are hereby incorporated by referencein their entirety for all purposes.

This application relates to U.S. application Ser. No. 11/043,895, filed14 Feb. 2005, which is incorporated by reference in its entirety for allpurposes.

BACKGROUND

Recently, there have been concerted efforts to develop and manufacturemicrofluidic systems to perform various chemical and biochemicalanalyses and syntheses, both for preparative and analyticalapplications. The goal to make such devices arises because of thesignificant benefits that can be realized from miniaturization withrespect to analyses and syntheses conducted on a macro scale. Suchbenefits include a substantial reduction in time, cost and the spacerequirements for the devices utilized to conduct the analysis orsynthesis. Additionally, microfluidic devices have the potential to beadapted for use with automated systems, thereby providing the additionalbenefits of further cost reductions and decreased operator errorsbecause of the reduction in human involvement. Microfluidic devices havebeen proposed for use in a variety of applications, including, forinstance, capillary electrophoresis, gas chromatography and cellseparations.

However, realization of these benefits has often been thwarted becauseof various complications associated with the microfluidic devices thathave thus far been manufactured. For instance, many of the currentmicrofluidic devices are manufactured from silica-based substrates;these materials are difficult and complicated to machine and devicesmade from such materials are fragile. Furthermore, transport of fluidthrough many existing microfluidic devices requires regulation ofcomplicated electrical fields to transport fluids in a controlledfashion through the device.

Thus, in view of the foregoing benefits that can be achieved withmicrofluidic devices but the current limitations of existing devices,there remains a need for microfluidic devices designed for use inconducting a variety of chemical and biochemical analyses. Because ofits importance in modern biochemistry, there is a particular need fordevices that can be utilized to conduct a variety of nucleic acidamplification reactions, while having sufficient versatility for use inother types of analyses as well.

Devices with the ability to conduct nucleic acid amplifications wouldhave diverse utilities. For example, such devices could be used as ananalytical tool to determine whether a particular target nucleic acid ofinterest is present or absent in a sample. Thus, the devices could beutilized to test for the presence of particular pathogens (e.g.,viruses, bacteria or fungi), and for identification purposes (e.g.,paternity and forensic applications). Such devices could also beutilized to detect or characterize specific nucleic acids previouslycorrelated with particular diseases or genetic disorders. When used asanalytical tools, the devices could also be utilized to conductgenotyping analyses and gene expression analyses (e.g., differentialgene expression studies). Alternatively, the devices can be used in apreparative fashion to amplify sufficient nucleic acid for furtheranalysis such as sequencing of amplified product, cell-typing, DNAfingerprinting and the like. Amplified products can also be used invarious genetic engineering applications, such as insertion into avector that can then be used to transform cells for the production of adesired protein product.

SUMMARY

A variety of devices and methods for conducting microfluidic analysesare provided herein, including devices that can be utilized to conductthermal cycling reactions such as nucleic acid amplification reactions.The devices differ from conventional microfluidic devices in that theyinclude elastomeric components; in some instances, much or all of thedevice is composed of elastomeric material.

Certain devices are designed to conduct thermal cycling reactions (e.g.,PCR) with devices that include one or more elastomeric valves toregulate solution flow through the device. Thus, methods for conductingamplification reactions with devices of this design are also provided.

Some of the devices include blind flow channels which include a regionthat functions as a reaction site. Certain such devices include a flowchannel formed within an elastomeric material, and a plurality of blindflow channels in fluid communication with the flow channel, with aregion of each blind flow channel defining a reaction site. The devicescan also include one or more control channels overlaying andintersecting each of the blind flow channels, wherein an elastomericmembrane separates the one or more control channels from the blind flowchannels at each intersection. The elastomeric membrane in such devicesis disposed to be deflected into or withdrawn from the blind flowchannel in response to an actuation force. The devices can optionallyfurther include a plurality of guard channels formed within theelastomeric material and overlaying the flow channel and/or one or moreof the reaction sites. The guard channels are designed to have fluidflow therethrough to reduce evaporation from the flow channels andreaction sites of the device. Additionally, the devices can optionallyinclude one or more reagents deposited within each of the reactionsites.

In certain devices, the flow channel is one of a plurality of flowchannels, each of the flow channels in fluid communication with multipleblind flow channels which branch therefrom. Of devices of this design,in some instances the plurality of flow channels are interconnected withone another such that fluid can be introduced into each of the reactionsites via a single inlet. In other devices, however, the plurality offlow channels are isolated from each other such that fluid introducedinto one flow channel cannot flow to another flow channel, and each flowchannel comprises an inlet at one or both ends into which fluid can beintroduced.

Other devices include an array of reaction sites having a density of atleast 50 sites/cm², with the reaction sites typically formed within anelastomeric material. Other devices have even higher densities such asat least 250, 500 or 1000 sites/cm², for example.

Still other device include a reaction site formed within an elastomericsubstrate, at which a reagent for conducting a reaction isnon-covalently immobilized. The reagent can be one or more reagents forconducting essentially any type of reaction. The reagent in some devicesincludes one reagents for conducting a nucleic acid amplificationreaction. Thus, in some devices the reagent comprises a primer,polymerase and one or more nucleotides. In other devices, the reagent isa nucleic acid template.

A variety of matrix or array-based devices are also provided. Certain ofthese devices include: (i) a first plurality of flow channels formed inan elastomeric substrate, (ii) a second plurality of flow channelsformed in the elastomeric substrate that intersect the first pluralityof flow channels to define an array of reaction sites, (iii) a pluralityof isolation valves disposed within the first and second plurality offlow channels that can be actuated to isolate solution within each ofthe reaction sites from solution at other reaction sites, and (iv) aplurality of guard channels overlaying one or more of the flow channelsand/or one or more of the reaction sites to prevent evaporation ofsolution therefrom.

The foregoing devices can be utilized to conduct a number of differenttypes of reactions, including those involving temperature regulation(e.g., thermocycling of nucleic acid analyses). Methods conducted withcertain blind channel type devices involve providing a microfluidicdevice that comprises a flow channel formed within an elastomericmaterial; and a plurality of blind flow channels in fluid communicationwith the flow channel, with an end region of each blind flow channeldefining a reaction site. At least one reagent is introduced into eachof the reaction sites, and then a reaction is detected at one or more ofthe reaction sites. The method can optionally include heating the atleast one reagent within the reaction site. Thus, for example, a methodcan involve introducing the components for a nucleic acid amplificationreaction and then thermocycling the components to form amplifiedproduct.

Other methods involve providing a microfluidic device comprising one ormore reaction sites, each reaction site comprising a first reagent forconducting an analysis that is non-covalently deposited on anelastomeric substrate. A second reagent is then introduced into the oneor more reaction sites, whereby the first and second reagents mix toform a reaction mixture. A reaction between the first and secondreagents at one or more of the reaction sites is subsequently detected.

Still other methods involve providing a microfluidic device comprisingan array of reaction sites formed within a substrate and having adensity of at least 50 sites/cm². At least one reagent is introducedinto each of the reaction sites. A reaction at one or more of thereaction sites is then detected.

Yet other methods involve providing a microfluidic device comprising atleast one reaction site which is formed within an elastomeric substrateand a plurality of guard channels also formed within the elastomericsubstrate. At least one reagent is introduced into each of the reactionsites and then heated within the reaction sites. A fluid is flowedthrough the guard channels before or during heating to reduceevaporation from the at least one reaction site. A reaction within theat least one reaction site is subsequently detected.

Additional devices designed to reduce evaporation of fluid from thedevice are also provided. In general, such devices comprise a cavitythat is part of a microfluidic network formed in an elastomericsubstrate; and a plurality of guard channels overlaying the cavity andseparated from the cavity by an elastomeric membrane. The guard channelin such devices is sized (i) to allow solution flow therethrough, and(ii) such that there is not a substantial reduction in solution flow in,out or through the cavity due to deflection of the membrane(s) uponapplication of an actuation force to the guard channels. Other suchdevices include (i) one or more flow channels and/or one or morereaction sites; and (ii) a plurality of guard channels overlaying themicrofluidic system and separated therefrom by elastomer, wherein thespacing between guard channels is between 1 μm to 1 mm. In other devicesthe spacing is between 5 μm and 500 μm, in other devices between 10 μmand 100 μm, and in still other devices between 40 μm and 75 μm.

In other embodiments of the invention provide for a microfluidic devicehave one or more sample channels having a plurality of valves incommunication therewith, wherein each sample channel, when filled with asample, can be partitioned into sub-samples for conducting analysis,such as amplification, for example, but not limited to PCR, includingTAQMAN™, and endpoint PCR, and isothermal amplification techniques, suchas INVADER™. Such microfluidic devices may have some sample channelsdevoted to partition and analyze known control samples, while othersample channels may be used to partition and analyze one or more testsamples. The arrangement of the separate sample channels may beinterdigitated or laid out as plots among the device surface, the latterbeing preferred for optimizing conditions for conducting analysis on aroutine basis. One advantage of partitioning a sample is to reduce theapparent concentration of a high background of wild type samplecontaining a low, such as one, two, three, four, five, or six orders ofmagnitude lower concentration of a mutant sample than the wild typesample. For example, when analyzing a sample containing a highconcentration of wild-type DNA that contains a very small, severalorders of magnitude, such as 10⁻³ less copies than the wild type. Bypartitioning the sample many-fold, such as by 100, 1,000, 10,000,100,000, or 1,000,000-fold, the ratio in each partition between wildtype and mutant DNA is changed from the original ratio such that thelikelihood of background PCR product that would be produced relative theamount of PCR product produced from the target mutant is minimized toyield a valid signal indicating the presence of the mutant DNA target inthe particular partitioned chamber. There may also be benefits affordedby conducting the reaction in very small volumes that are obtainable incertain embodiments of the present invention, in that by conducting thereaction in a small volume raises the concentration of target to thevolume of the total reaction.

In another embodiment, the invention provides a method for analyzing thepresence of a specific gene, for example but not limited to an oncogenepoint mutation in a patient suspected of having a tumor if the tumorreleases genetic material into the body, in particular, the bloodstream, even though other non-oncogenic genetic material may be presentin the blood stream in great excess, including one, two, three, four,five, and six orders of magnitude greater excess than the targetoncogenic target genetic material. For example, in the K-ras pointmutation at codon 12 occurs in about 70 to 95% of the cells of thiscancer, wherein some or all of the cells may release genetic materialcontaining the K-ras point mutation at codon 12 which can be detected bythe methods described herein using the devices herein. In yet anotherembodiment, a sample may contain whole cells that when analyzed, forexample but not limited to, by PCR, the cells lyse and make theirgenetic material available for amplification. Other oncogenic pointmutations and genetic diseases that produce amplifiable genetic materialin low quantities relative to the background genetic material normallypresent

Compositions for conducting nucleic acid analyses in reaction sites ofcertain microfluidic devices are also provided. Certain suchcompositions include one or more of the following: an agent that blocksprotein binding sites on an elastomeric material and a detergent. Theblocking agent is typically selected from the group consisting of aprotein (e.g., gelatin or albumin, such as bovine serum albumin (BSA)).The detergent can be SDS or Triton, for example

In a particular application the present invention encompasses devicesand methods used for highly sensitive and specific detection of mutantalleles, particularly in the presence of a very high background ofwild-type genes. In particular, the present invention describes methodsthat can be used to specifically and reliably detect a single copy of amutant gene (for example, but not limited to BCR-Abl and K-ras mutants)in a sample containing about 10⁵ wild-type copies of the gene.

Using the present invention a single copy of a mutant target may bereliably detected in a background of wherein the ratio of the target tothe non-specific polynucleotides is, for example about 1:1×10³, 1:1×10⁴,1:1×10⁵, 1:1×10⁶, 1:1×10⁷, 1:1×10⁸, 1:1×10⁹, 1:1×10¹⁰ or even withhigher ratios.

The present invention additionally encompasses systems for performingmassive partitioning of a sample followed by PCR, followed by detectionof a PCR product. Such systems include any combination of elementsdescribed herein, for example, a microfluidic device having one or moreelastomeric valve, a sample input in fluid communication with at leastone flow channel that can be partitioned into a plurality of isolatedreaction chambers. The system further comprises a heat-exchange plate incontact with the microfluidic device for performing thermocyclingreactions. The system may optionally include a reader (e.g., afluorescent reader) for detecting and reading the results of a pluralityof PCR reactions.

The present invention additionally encompasses the use of a microfluidicdevice for performing massive partitioning of a sample followed by PCR,followed by detection of a PCR product. In particular, the inventionencompasses the use of a microfluidic device to separate a sample intoat least 1000, 2000, 3000, 4000, 5000, 10000, 20000 or 50000 separatesamples, each of which can be used for the performance of a PCRreaction.

The present invention additionally encompasses the use of a microfluidicsystem for performing massive partitioning of a sample followed by PCR,followed by detection of a PCR product, the system comprisingmicrofluidic device having one or more elastomeric valve, a sample inputin fluid communication with at least one flow channel that can bepartitioned into a plurality of isolated reaction chambers. The systemfurther comprises a heat-exchange plate in contact with the microfluidicdevice. The system may optionally include a reader for reading theresults of a plurality of PCR reactions.

The present invention additionally encompasses kits for performingmassive partitioning of a sample followed by PCR, followed by detectionof a PCR product, which kits may include any combination of elementsdescribed herein, for example, a microfluidic device having one or moreelastomeric valve, a sample input in fluid communication with at leastone flow channel that can be partitioned into a plurality of isolatedreaction chambers, a heat-exchange plate in contact with themicrofluidic device, and optionally, a reader for detecting and readingthe results of a plurality of PCR reactions.

The present invention further encompass a reader for reading a pluralityof PCR reactions performed using the devices described herein. Thereader may include a plate-holder for retaining the SBS-formatted deviceof the invention and one or more optical sensors adapted to detect afluorescent signal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of an exemplary device with amatrix design of intersecting vertical and horizontal flow channels.

FIGS. 1B-E show enlarged views of a portion of the device shown in FIG.1A and illustrate its operation.

FIG. 1F is a schematic representation of another exemplary matrix designdevice that utilizes guard channels to reduce sample evaporation.

FIG. 2 is a plan view of an exemplary blind channel device. The device200 includes a flow channel 204 and a set of branch flow channels 206branching therefrom that are formed in an elastomeric substrate 202.Overlaying the branch flow channels 206 is a control channel 210 that isseparated from the branch flow channels 206 by membranes 212.

FIG. 3A is a plan view of another exemplary blind channel device. Acontrol channel 310 overlays each of the branch channels 36 and isseparated from the underlying branch channel by membrane 312.

FIG. 3B is a schematic representation of a more complex blind channeldevice based upon the unit of the general design depicted in FIG. 3A.During operation, water is flowed through the guard channels 320 duringheating of the device 300 to increase the local concentration of waterin the device, thereby reducing evaporation of water from solution inthe flow channels 306 and reaction sites 308.

FIG. 4 is a plan view of a device utilizing the hybrid design. Actuationof the control channel at inlet 416 causes deflection of membranes 412into the branch flow channels 406 and isolation of reaction sites 408.

FIG. 5 is a chart showing ramp up and down times to conduct athermocycling reaction. It illustrates the rapidity at which the desiredtemperature is achieved using a blind channel device.

FIG. 6 shows the location of spotted reagents within reaction sites in ablind channel type device illustrating proper alignment of the reagentswithin reaction sites at the corners of the device.

FIGS. 7A and 7B respectively are a cross-sectional view and a schematicdiagram of another hybrid type microfluidic device and represents thetype of device used to conduct the experiments described in Examples1-4.

FIG. 8 is a bar graph in which the average FAM/PR1/Control ratios areplotted for six different β-actin TaqMan reactions. The reactions werethermocycled in the micro fluidic device (chip) shown in FIG. 7B (solidbars) and Macro TaqMan reactions (striped bars). The controls are thefirst and fourth bar sets that have no DNA. The error bars are thestandard deviation of the ratios.

FIG. 9 is a diagram depicting an exemplary pin spotting process.Reagents are picked up from a source (e.g., a microtiter plate) and thenprinted by bringing the loaded pin into contact with the substrate. Thewash step consists of agitation in deionized water followed by vacuumdrying.

FIG. 10 is a bar graph depicting FAM signal strength for themicrofluidic device (chip) described in Example 1 (see FIG. 7B) based onthe experiments described in Example 2. The data are in the form of (FAMsignal/PR1 signal) scaled by the FAM/PR1 ratio for the reference lanes.Error bars are the standard deviation along a lane. The “1.3×” and “1×”designations refer to the concentration of the spotted primers andprobes, in relation to their nominal values.

FIG. 11 is a bar graph showing average VIC/PF1/Control ratios for 9-10wells for Macro TaqMan (striped bars), and TaqMan reactions in themicrofluidic device (solid bars). Two negative controls (Control) andtwo samples with 100 pg/nl genomic DNA were thermocycled with reactioncomponents as described above with 4× the standard amount ofprimer/probe. The error bars represent the standard deviation of theaverage ratios.

FIG. 12 is a bar graph that shows FAM/PR1/Control ratios for each of10-1 nl wells branching from a single flow channel of a microfluidicdevice (see FIG. 7B). The amount of genomic DNA was 0.25 pg/nl, whichresults in an average of one target copy per well.

FIG. 13 is a bar graph depicting the average VIC/PR1/Control ratios forCYP2D6 SNP reactions using the microfluidic device shown in FIG. 7B.Allele 1 (Al-1) is the positive control for the VIC probe against thereference or wild type allele CYP2D6*1. Allele 2 (Al-2) is the positivecontrol for the FAM probe against the variant or mutant allele,CYP2D6*3. The control has no DNA template. Genomic DNA was used ateither 100 pg/nl or 20 pg/nl. The error bars are the standard deviationof the ratios.

FIG. 14 is a bar graph showing the average FAM/PR1/Control ratios forCYP2D6 SNP reactions in the microfluidic device shown in FIG. 7B. Thesamples are the same as described with respect to FIG. 13 and in Example3.

FIG. 15 is a schematic diagram of the microfluidic device used for theexperiments in Example 4. In this example, the reaction sites, depictedas square boxes along the serpentine, fluid channel, would contain aplurality of different SNP probes.

FIG. 16 is a polyacrylamide gel containing PCR product from Macro PCRand PCR reactions in the microfluidic device shown in FIG. 7B. Theresults on the left show the approximate migration of different DNA basepair lengths. The lanes containing interspersed bands are molecularweight markers. The lanes labeled “Macro” are the PCR products from theMacro reactions at different dilutions. The lanes labeled “In chip” arePCR products generated in the chip. The lanes containing many bandsthroughout the gel are nonspecific background signals.

FIGS. 17 a-17 d depict two preferred designs of a partitioningmicrofluidic device in a valve off and valve actuated state. Multilayersoft lithography (SL) (1), was used to create elastomeric microfluidicchips which use active valves to partition each of several liquidsamples into a multitude of isolated reaction volumes.

FIGS. 18 a and 18 b depict images of a partitioning microfluidic devicesafter a thermocycling reaction was performed. FIG. 18 a is ablack-and-white depiction of a two color image, and FIG. 18 b depictsthe remaining signal after subtraction of the control red signal.

FIG. 19 depicts a graph of comparing the average number of copies perwell to the number of positive wells. The number of positive wells wasconsistent with the number of wells calculated to have greater than orequal to 1 copy of target by the Poisson distribution.

FIG. 20 depicts an isothermic amplification scheme referred to as“Scorpion”.

FIG. 21 depicts an exemplary matrix microfluidic device plan view. Byhaving a fluid line in each layer of a two layer elastomeric block,higher density reaction cell arrangements are possible.

FIGS. 22, 23, and 24 depict detection of rare targets among highbackground genetic material samples.

FIG. 25 is a schematic diagram of the BCR-Abl tyrosine kinase domainmutations and exploded view of exons 4 and 6, with primers and probesused in PCR assays indicated.

FIG. 26 shows a fluorescent image of the DID chip after 32 cycles ofmutation-specific PCR assay for Q252H G>C and Q252H G>T. TaqManreactions contained mutant-specific primers and Black Hole Quencher®probe with Cal-Fluor Orange® reporter. Other mutation-specific PCRassays were developed for the following mutations: G250E, Y253F, E255K,E255V, and T315I.

FIGS. 27A and 27B shows images of DID chips after indicated cycles ofmutation-specific PCR assays for T315I and Y253F. Single mutant cDNAcopies were detected in background of up to 10⁵ WT BCR-Abl cDNA copies.A) T315I-specific TaqMan reactions contained mutant-specific primers,Black Hole Quencher® probe with 6-FAM reporter. B) Y253F-specific TaqManreactions contained mutant-specific primers, Black Hole Quencher Probe®with Cal-Fluor Orange® reporter.

FIGS. 28A and 28B show fluorescent images of DID chip after 39 cycles ofT315I-Specific PCR and Total BCR-Abl-Specific PCR assays. Patient totalrandom-primed cDNA prepared from leukocytes. Positive control mutantBCR-Abl cDNA from BaF3 cells prepared by BCR-Abl-specific reversetranscription reaction. A) T315I Mutant-specific TaqMan PCR on DID chip:single mutant copies detected and quantified as indicated in complexbackground of greater than 10³ BCR-Abl copies. B) Total BCR-Ablamplified with primers exterior to codon 315 by TaqMan PCR on DID chip:Saturated signal points out greater than 10³ copies. Sample dilutionswere required for absolute quantification of total BCR-Abl.

DETAILED DESCRIPTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

A “flow channel” refers generally to a flow path through which asolution can flow.

The term “valve” unless otherwise indicted refers to a configuration inwhich a flow channel and a control channel intersect and are separatedby an elastomeric membrane that can be deflected into or retracted fromthe flow channel in response to an actuation force.

A “blind channel” or a “dead-end channel” refers to a flow channel whichhas an entrance but not a separate exit. Accordingly, solution flow inand out of the blind channel occurs at the same location. The process offilling one or more blind channels is sometimes simply referred to as“blind fill.”

An “isolated reaction site” generally refers to a reaction site that isnot in fluid communication with other reactions sites present on thedevice. When used with respect to a blind channel, the isolated reactionsite is the region at the end of the blind channel that can be blockedoff by a valve associated with the blind channel.

A “via” refers to a channel formed in an elastomeric device to providefluid access between an external port of the device and one or more flowchannels. Thus, a via can serve as a sample input or output, forexample.

The term “elastomer” and “elastomeric” has its general meaning as usedin the art. Thus, for example, Allcock et al. (Contemporary PolymerChemistry, 2nd Ed.) describes elastomers in general as polymers existingat a temperature between their glass transition temperature andliquefaction temperature. Elastomeric materials exhibit elasticproperties because the polymer chains readily undergo torsional motionto permit uncoiling of the backbone chains in response to a force, withthe backbone chains recoiling to assume the prior shape in the absenceof the force. In general, elastomers deform when force is applied, butthen return to their original shape when the force is removed. Theelasticity exhibited by elastomeric materials can be characterized by aYoung's modulus. The elastomeric materials utilized in the microfluidicdevices disclosed herein typically have a Young's modulus of betweenabout 1 Pa-1 TPa, in other instances between about 10 Pa-100 GPa, instill other instances between about 20 Pa-1 GPa, in yet other instancesbetween about 50 Pa-10 MPa, and in certain instances between about 100Pa-1 MPa. Elastomeric materials having a Young's modulus outside ofthese ranges can also be utilized depending upon the needs of aparticular application.

Some of the microfluidic devices described herein are fabricated from anelastomeric polymer such as GE RTV 615 (formulation), a vinyl-silanecrosslinked (type) silicone elastomer (family). However, the presentmicrofluidic systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a large number of possible elastomer systems thatcan be used to make monolithic elastomeric microvalves and pumps. Thechoice of materials typically depends upon the particular materialproperties (e.g., solvent resistance, stiffness, gas permeability,and/or temperature stability) required for the application beingconducted. Additional details regarding the type of elastomericmaterials that can be used in the manufacture of the components of themicrofluidic devices disclosed herein are set forth in Unger et al.(2000) Science 288:113-116, and PCT Publications WO 02/43615, and WO01/01025, which are incorporated herein by reference in their entiretyfor all purposes.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused herein to include a polymeric form of nucleotides of any length,including, but not limited to, ribonucleotides or deoxyribonucleotides.There is no intended distinction in length between these terms. Further,these terms refer only to the primary structure of the molecule. Thus,in certain embodiments these terms can include triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. They also include modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,”include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing normucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA.

A “probe” is a nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target. The labelattached to the probe can include any of a variety of different labelsknown in the art that can be detected by chemical or physical means, forexample. Suitable labels that can be attached to probes include, but arenot limited to, radioisotopes, fluorophores, chromophores, mass labels,electron dense particles, magnetic particles, spin labels, moleculesthat emit chemiluminescence, electrochemically active molecules,enzymes, cofactors, and enzyme substrates. Probes can vary significantlyin size. Some probes are relatively short. Generally, probes are atleast 7 to 15 nucleotides in length. Other probes are at least 20, 30 or40 nucleotides long. Still other probes are somewhat longer, being atleast 50, 60, 70, 80, 90 nucleotides long. Yet other probes are longerstill, and are at least 100, 150, 200 or more nucleotides long. Probescan be of any specific length that falls within the foregoing ranges aswell.

A “primer” is a single-stranded polynucleotide capable of acting as apoint of initiation of template-directed DNA synthesis under appropriateconditions (i.e., in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature. The appropriate length of a primer depends on theintended use of the primer but typically is at least 7 nucleotides longand, more typically range from 10 to 30 nucleotides in length. Otherprimers can be somewhat longer such as 30 to 50 nucleotides long. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. The term “primer site” or“primer binding site” refers to the segment of the target DNA to which aprimer hybridizes. The term “primer pair” means a set of primersincluding a 5′ “upstream primer” that hybridizes with the complement ofthe 5′ end of the DNA sequence to be amplified and a 3′ “downstreamprimer” that hybridizes with the 3′ end of the sequence to be amplified.

A primer that is “perfectly complementary” has a sequence fullycomplementary across the entire length of the primer and has nomismatches. The primer is typically perfectly complementary to a portion(subsequence) of a target sequence. A “mismatch” refers to a site atwhich the nucleotide in the primer and the nucleotide in the targetnucleic acid with which it is aligned are not complementary. The term“substantially complementary” when used in reference to a primer meansthat a primer is not perfectly complementary to its target sequence;instead, the primer is only sufficiently complementary to hybridizeselectively to its respective strand at the desired primer-binding site.

The term “complementary” means that one nucleic acid is identical to, orhybridizes selectively to, another nucleic acid molecule. Selectivity ofhybridization exists when hybridization occurs that is more selectivethan total lack of specificity. Typically, selective hybridization willoccur when there is at least about 55% identity over a stretch of atleast 14-25 nucleotides, preferably at least 65%, more preferably atleast 75%, and most preferably at least 90%. Preferably, one nucleicacid hybridizes specifically to the other nucleic acid. See M. Kanehisa,Nucleic Acids Res. 12:203 (1984).

The term “label” refers to a molecule or an aspect of a molecule thatcan be detected by physical, chemical, electromagnetic and other relatedanalytical techniques. Examples of detectable labels that can beutilized include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, enzymes linked to nucleic acidprobes and enzyme substrates. The term “detectably labeled” means thatan agent has been conjugated with a label or that an agent has someinherent characteristic (e.g., size, shape or color) that allows it tobe detected without having to be conjugated to a separate label.

A “polymorphic marker” or “polymorphic site” is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphic locus may be assmall as one base pair. Polymorphic markers include restriction fragmentlength polymorphisms, variable number of tandem repeats (VNTR's),hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats, simple sequence repeats,and insertion elements such as Alu. The first identified allelic form isarbitrarily designated as the reference form and other allelic forms aredesignated as alternative or variant alleles. The allelic form occurringmost frequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

A “single nucleotide polymorphism” (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A singlenucleotide polymorphism usually arises due to substitution of onenucleotide for another at the polymorphic site. A transition is thereplacement of one purine by another purine or one pyrimidine by anotherpyrimidine. A transversion is the replacement of a purine by apyrimidine or vice versa. Single nucleotide polymorphisms can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele.

A “reagent” refers broadly to any agent used in a reaction. A reagentcan include a single agent which itself can be monitored (e.g., asubstance that is monitored as it is heated) or a mixture of two or moreagents. A reagent may be living (e.g., a cell) or non-living. Exemplaryreagents for a nucleic acid amplification reaction include, but are notlimited to, buffer, metal ions, polymerase, primers, template nucleicacid, nucleotides, labels, dyes, nucleases and the like. Reagents forenzyme reactions include, for example, substrates, cofactors, couplingenzymes, buffer, metal ions, inhibitors and activators. Reagents forcell-based reactions include, but are not limited to, cells, cellspecific dyes and ligands (e.g., agonists and antagonists) that bind tocellular receptors.

A “ligand” is any molecule for which there exists another molecule(i.e., an “antiligand”) that specifically or non-specifically binds tothe ligand, owing to recognition of some portion of the ligand by theantiligand.

Non-specific polynucleotides are polynucleotides other than the specifictarget, e.g., genomic DNA and or wild-type DNA.

When “DNA” is used herein, it is implied that it RNA may equally be usedin its place in most instances, as will be recognized by one of skill inthe art.

II. Overview

A number of different microfluidic devices (also sometimes referred toas chips) having unique flow channel architectures are provided herein,as well as methods for using such devices to conduct a variety of highthroughput analyses. The devices are designed for use in analysesrequiring temperature control, especially analyses involvingthermocycling (e.g., nucleic acid amplification reactions). Themicrofluidic devices incorporate certain design features that: give thedevices a significantly smaller footprint than many conventionalmicrofluidic devices, enable the devices to be readily integrated withother instrumentation and provide for automated analysis.

Some of the microfluidic devices utilize a design typically referred toherein as “blind channel” or “blind fill” are characterized in part byhaving a plurality of blind channels, which, as indicated in thedefinition section, are flow channels having a dead end or isolated endsuch that solution can only enter and exit the blind channel at one end(i.e., there is not a separate inlet and outlet for the blind channel).These devices require only a single valve for each blind channel toisolate a region of the blind channel to form an isolated reaction site.During manufacture of this type of device, one or more reagents forconducting an analysis are deposited at the reaction sites, therebyresulting in a significant reduction in the number of input and outputs.Additionally, the blind channels are connected to an interconnectednetwork of channels such that all the reaction sites can be filled froma single, or limited number, of sample inputs. Because of the reductionin complexity in inputs and outputs and the use of only a single valveto isolate each reaction site, the space available for reaction sites isincreased. Thus, the features of these devices means that each devicecan include a large number of reaction sites (e.g., up to tens ofthousands) and can achieve high reaction site densities (e.g., over1,000-4,000 reaction sites/cm²). Individually and collectively, thesefeatures also directly translate into a significant reduction in thesize of these devices compared to traditional microfluidic devices.

Other microfluidic devices that are disclosed herein utilize a matrixdesign. In general, microfluidic devices of this type utilize aplurality of intersecting horizontal and vertical flow channels todefine an array of reaction sites at the points of intersection. Thus,devices of this design also have an array or reaction sites; however,there is a larger number of sample inputs and corresponding outputs toaccommodate the larger number of samples with this design. A valvesystem referred to as a switchable flow array architecture enablessolution be flowed selectively through just the horizontal or flowchannels, thus allowing for switchable isolation of various flowchannels in the matrix. Hence, whereas the blind channel devices aredesigned to conduct a large number of analyses under differentconditions with a limited number of samples, the matrix devices areconstructed to analyze a large number of sample under a limited numberof conditions. Still other devices are hybrids of these two generaldesign types.

The microfluidic devices that are described are further characterized inpart by utilizing various components such as flow channels, controlchannels, valves and/or pumps from elastomeric materials. In someinstances, essentially the entire device is made of elastomericmaterial. Consequently, such devices differ significantly in form andfunction from the majority of conventional microfluidic devices that areformed from silicon-based material.

The design of the devices enables them to be utilized in combinationwith a number of different heating systems. Thus, the devices are usefulin conducting diverse analyses that require temperature control.Additionally, those microfluidic devices for use in heating applicationscan incorporate a further design feature to minimize evaporation ofsample from the reaction sites. Devices of this type in general includea number of guard channels formed within the elastomeric device throughwhich water can be flowed to increase the water vapor pressure withinthe elastomeric material from which the device is formed, therebyreducing evaporation of sample from the reaction sites.

In another embodiment, a temperature cycling device may be used tocontrol the temperature of the microfluidic devices. Preferably, themicrofluidic device would be adapted to make thermal contact with themicrofluidic device. Where the microfluidic device is supported by asubstrate material, such as a glass slide or the bottom of a carrierplate, such as a plastic carrier, a window may be formed in a region ofthe carrier or slide such that the microfluidic device, preferably adevice having an elastomeric block, may directly contact theheating/cooling block of the temperature cycling device. In a preferredembodiment, the heating/cooling block has grooves therein incommunication with a vacuum source for applying a suction force to themicrofluidic device, preferably the portion wherein the reactions aretaking place. Alternatively, a rigid thermally conductive plate may bebonded to the microfluidic device that then mates with the heating andcooling block for efficient thermal conduction resulting.

The array format of certain of the devices means the devices can achievehigh throughput. Collectively, the high throughput and temperaturecontrol capabilities make the devices useful for performing largenumbers of nucleic acid amplifications (e.g., polymerase chainreaction—PCR). Such reactions will be discussed at length herein asillustrative of the utility of the devices, especially of their use inany reaction requiring temperature control. However, it should beunderstood that the devices are not limited to these particularapplications. The devices can be utilized in a wide variety of othertypes of analyses or reactions. Examples include analyses ofprotein-ligand interactions and interactions between cells and variouscompounds. Further examples are provided infra.

III. General Structure of Microfluidic Devices

A. Pumps and Valves

The microfluidic devices disclosed herein are typically constructed atleast in part from elastomeric materials and constructed by single andmultilayer soft lithography (MSL) techniques and/or sacrificial-layerencapsulation methods (see, e.g., Unger et al. (2000) Science288:113-116, and PCT Publication WO 01/01025, both of which areincorporated by reference herein in their entirety for all purposes).Utilizing such methods, microfluidic devices can be designed in whichsolution flow through flow channels of the device is controlled, atleast in part, with one or more control channels that are separated fromthe flow channel by an elastomeric membrane or segment. This membrane orsegment can be deflected into or retracted from the flow channel withwhich a control channel is associated by applying an actuation force tothe control channels. By controlling the degree to which the membrane isdeflected into or retracted out from the flow channel, solution flow canbe slowed or entirely blocked through the flow channel. Usingcombinations of control and flow channels of this type, one can preparea variety of different types of valves and pumps for regulating solutionflow as described in extensive detail in Unger et al. (2000) Science288:113-116, and PCT Publication WO/02/43615 and WO 01/01025.

The devices provided herein incorporate such pumps and valves to isolateselectively a reaction site at which reagents are allowed to react. Thereaction sites can be located at any of a number of different locationswithin the device. For example, in some matrix-type devices, thereaction site is located at the intersection of a set of flow channels.In blind channel devices, the reaction site is located at the end of theblind channel.

If the device is to be utilized in temperature control reactions (e.g.,thermocycling reactions), then, as described in greater detail infra,the elastomeric device is typically fixed to a support (e.g., a glassslide). The resulting structure can then be placed on a temperaturecontrol plate, for example, to control the temperature at the variousreaction sites. In the case of thermocycling reactions, the device canbe placed on any of a number of thermocycling plates.

Because the devices are made of elastomeric materials that arerelatively optically transparent, reactions can be readily monitoredusing a variety of different detection systems at essentially anylocation on the microfluidic device. Most typically, however, detectionoccurs at the reaction site itself (e.g., within a region that includesan intersection of flow channels or at the blind end of a flow channel).The fact that the device is manufactured from substantially transparentmaterials also means that certain detection systems can be utilized withthe current devices that are not usable with traditional silicon-basedmicrofluidic devices. Detection can be achieved using detectors that areincorporated into the device or that are separate from the device butaligned with the region of the device to be detected.

B. Guard Channels

To reduce evaporation of sample and reagents from the elastomericmicrofluidic devices that are provided herein, a plurality of guardchannels can be formed in the devices. The guard channels are similar tothe control channels in that typically they are formed in a layer ofelastomer that overlays the flow channels and/or reaction site. Hence,like control channels, the guard channels are separated from theunderlying flow channels and/or reaction sites by a membrane or segmentof elastomeric material. Unlike control channels, however, the guardchannels are considerably smaller in cross-sectional area. In general, amembrane with smaller area will deflect less than a membrane with largerarea under the same applied pressure. The guard channels are designed tobe pressurized to allow solution (typically water) to be flowed into theguard channel. Water vapor originating from the guard channel candiffuse into the pores of the elastomer adjacent a flow channel orreaction site, thus increasing the water vapor concentration adjacentthe flow channel or reaction site and reducing evaporation of solutiontherefrom.

In general, the guard channels are sufficiently small such that whenpressurized the membrane that separates the guard channel from theunderlying flow channel or reaction site does not substantially restrictsolution flow in, out, or through the flow channel or reaction sitewhich the guard channel overlays. When used in this context, the term“substantially restrict” or other similar terms means that solution flowis not reduced in, out or through the flow channel or reaction site bymore than 40%, typically less than 30%, usually less than 20%, and insome instances less than 10%, as compared to solution flow in, to orthrough the flow channel or reaction site under the same conditions,when the guard channel is not pressurized to achieve solution flowtherethrough. Usually this means that the guard channels have across-sectional area of between 100 μm² and 50,000 μm², or any integralor non-integral cross-sectional area therebetween. Thus, for example, insome instances, the cross-sectional area is less than 50,000 μm², inother instances less than 10,000 μm², in still other instances less than10,00 μm², and in yet other instances less than 100 μm². The guardchannels can have any of a variety of shapes including, but not limitedto, circular, elliptical, square, rectangular, hexagonal and octahedralshapes.

The guard channels are designed to reduce the evaporation of sample andreagents from the device during the time and under the conditions thatit takes to conduct a thermocycling reaction to less than 50%, in otherinstance less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1%.Thus, for example, a typical PCR reaction involving 40 cycles can beconducted within 120 minutes. The guard channel system is designed toreduce evaporation during approximately this time frame to the foregoingset of limits. To achieve this level of evaporation reduction, the guardchannels are typically present at a density of at least 10 lines/cm² to1000 lines/cm², or any integral density level therebetween. Morespecifically, the guard channels are generally at least 25 lines/cm², inother instances at least 50 lines/cm², in still other instances at least100 lines/cm², and in yet other instances at least 500 lines/cm². Toachieve this level of evaporation reduction, the guard channels aretypically present at a spacing between 1 mm to 1 μm as measured from theouter edge of one line to the nearest outer edge of adjacent line, orany integral density level therebetween. More specifically, the guardchannels are generally spaced between 500 μm to 5 μm, in other instancesbetween 100 μm to 10 μm, in still other instances between 75 μm to 40μm. Thus, the spacing is typically at least 1 μm, but is less than 1 mm,in other instances less than 500 μm, in still other instances less than400 μm, in yet other instances less than 300 μm, in other instances lessthan 200 μm, and in still other instances less than 100 μm, 50 μm or 25μm.

The guard channels can be formed as a separate network of channels orcan be smaller channels that branch off of the control channels. Theguard channels can extend across the device or only a particular regionor regions of the device. Typically, the guard channels are placedadjacent and over flow channels and reaction sites as these are theprimary locations at which evaporation is the primary concern. Exemplarylocations of guard channels on certain matrix devices are illustrated inFIG. 1C, and on certain blind channel devices in FIG. 3B, and discussedin greater detail infra.

The solution flowed through the guard channel includes any substancethat can reduce evaporation of water. The substance can be one thatincreases the water vapor concentration adjacent a flow line and/orreaction site, or one that while not increasing the water vaporconcentration nonetheless blocks evaporation of water from the flow lineand/or reaction site (blocking agent). Thus, one option is to utilizeessentially any aqueous solution in which case suitable solutionsinclude, but are not limited to, water and buffered solution (e.g.,TaqMan buffer solution, and phosphate buffered saline). Suitableblocking agents include, for example, mineral oil.

Guard channels are typically formed in the elastomer utilizing the MSLtechniques and/or sacrificial-layer encapsulation methods cited above.

The following sections describe in greater detail a number of specificconfigurations that can be utilized to conduct a variety of analyses,including analyses requiring temperature control (e.g., nucleic acidamplification reactions). It should be understood, however, that theseconfigurations are exemplary and that modifications of these systemswill be apparent to those skilled in the art.

IV. Matrix Design

A. General

Devices utilizing the matrix design generally have a plurality ofvertical and horizontal flow channel that intersect to form an array ofjunctions. Because a different sample and reagent (or set of reagents)can be introduced into each of the flow channels, a large number ofsamples can be tested against a relatively large number of reactionconditions in a high throughput format. Thus, for example, if adifferent sample is introduced into each of M different vertical flowchannels and a different reagent (or set of reagents) is introduced intoeach of N horizontal flow channels, then M×N different reactions can beconducted at the same time. Typically, matrix devices include valvesthat allow for switchable isolation of the vertical and horizontal flowchannels. Said differently, the valves are positioned to allow selectiveflow just through the vertical flow channels or just through thehorizontal flow channels. Because devices of this type allow flexibilitywith respect to the selection of the type and number of samples, as wellas the number and type of reagents, these devices are well-suited forconducting analyses in which one wants to screen a large number ofsamples against a relatively large number of reaction conditions. Thematrix devices can optionally incorporate guard channels to help preventevaporation of sample and reactants.

The invention provides for high-density matrix designs that utilizefluid communication vias between layers of the microfluidic device toweave control lines and fluid lines through the device. For example, byhaving a fluid line in each layer of a two layer elastomeric block,higher density reaction cell arrangements are possible. FIG. 21 depictsan exemplary matrix design wherein a first elastomeric layer (1st layer)and a second elastomeric layer (2d layer) each having fluid channelsformed therein. For example, a reagent fluid channel in the first layeris connected to a reagent fluid channel in the second layer through avia, while the second layer also has sample channels therein, the samplechannels and the reagent channels terminating in sample and reagentchambers, respectively. The sample and reagent chambers are in fluidcommunication with each other through an interface channel that has aninterface valve associated therewith to control fluid communicationbetween each of the chambers of a reaction cell. In use, the interfaceis first closed, then reagent is introduced into the reagent channelfrom the reagent inlet and sample is introduced into the sample channelthrough the sample inlet, containment valves are then closed to isolateeach reaction cell from other reaction cells. Once the reaction cellsare isolated, the interface valve is opened to cause the sample chamberand the reagent chamber to be in fluid communication with each other sothat a desired reaction may take place.

Accordingly, a preferred aspect of the invention provides for amicrofluidic device for reacting M number of different samples with Nnumber of different reagents comprising: a plurality of reaction cells,each reaction cell comprising a sample chamber and a reagent chamber,the sample chamber and the reagent chamber being in fluid communicationthrough an interface channel having an interface valve associatedtherewith for controlling fluid communication between the sample chamberand the reagent chamber; a plurality of sample inlets each in fluidcommunication with the sample chambers; a plurality of reagent inletseach in fluid communication with the reagent chambers; wherein one ofthe sample inlets or reagent inlets is in fluid communication with oneof the sample chambers or one of the reagent chambers, respectively,through a via. Certain embodiments include having the reaction cells beformed within an elastomeric block formed from a plurality of layersbonded together and the interface valve is deflectable membrane; havingthe sample inlets be in communication with the sample chamber through asample channel and the reagent inlet is in fluid communication with thereagent chamber through a reagent channel, a portion of the samplechannel and a portion of the reagent channel being oriented aboutparallel to each other and each having a containment valve associatedtherewith for controlling fluid communication therethrough; having thevalve associated with the sample channel and the valve associated withthe reagent channel are in operable communication with each otherthrough a common containment control channel; having the containmentcommon control channel located along a line about normal to one of thesample channel or the reagent channel

Another aspect of the invention provides for a method for making afeature in an elastomeric block comprising the steps of: providing afirst elastomeric layer; applying a photoresist layer upon a surface ofthe first elastomeric layer; applying a light pattern to the photoresistlayer to form a pattern of reacted photoresist material; removingunreacted photoresist material leaving the pattern of reactedphotoresist upon the surface of the first elastomeric layer; applying anetching reagent to the first elastomeric surface to etch the surface ofthe first elastomeric layer not covered by the pattern of reactedphotoresist material thereby removing regions of the first elastomericlayer not covered by the pattern of reacted photoresist and leaving apattern of the elastomeric layer corresponding to the pattern of reactedphotoresist material. In certain preferred embodiments of the methodinclude having a step of removing the pattern of reacted photoresistmaterial; having the removing is caused by applying an adhesive tape tothe surface of the elastomeric layer and the pattern of reactedphotoresist material, then separating the adhesive tape from theelastomeric layer while some or all of the pattern of reactedphotoresist material is removed from the surface of the elastomericlayer; having the photo resist be SU8; having the etching reagentcomprises tetrabutylammoniumfluoride-trihydrate; having the feature be avia; having the elastomeric block comprise a plurality of elastomericlayers bonded together, wherein two or more elastomeric layers haverecesses formed therein and one recess of one elastomeric layer is incommunication with a recess of another elastomeric layer through thevia.

The microfluidic devices of the present invention may be furtherintegrated into the carrier devices described in copending and co-ownedU.S. patent application Ser. No. 60/557,715 by Unger filed on Mar. 29,2004, which is herein incorporated for all purposes. The carrier ofUnger provides on-board continuous fluid pressure to maintain valveclosure away from a source of fluid pressure, e.g., house air pressure.Unger further provides for an automated system for charging andactuating the valves of the present invention as described therein.

B. Exemplary Designs and Uses

FIG. 1A provides an illustration of one exemplary matrix device. Thisdevice 100 comprises seven vertical flow channels 102 and sevenhorizontal flow channels 104 that intersect to form an array of 49different intersections or reaction sites 106. This particular devicethus enables seven samples to be reacted with seven different reagentsor sets of reagents. Column valves 110 that regulate solution flow inthe vertical direction can be controlled by control channels 118 thatcan all be actuated at a single inlet 114.

Similarly, row valves 108 regulate solution flow in the horizontaldirection; these are controlled by control channels 116 that areactuated by a single control inlet 112. As shown in FIG. 1A, the controlchannels 116 that regulate the row valves 108 vary in width dependingupon location. When a control channel 116 crosses a vertical flowchannel 102, the control channel 116 is sufficiently narrow that when itis actuated it does not deflect into the vertical flow channel 102 toreduce substantially solution flow therethrough. However, the width ofthe control channel 116 is increased when it overlays one of thehorizontal flow channels 104; this makes the membrane of the controlchannel sufficiently large to block solution flow through the horizontalflow channel 104.

In operation, reagents R1-R7 are introduced into their respectivehorizontal flow channels 104 and samples S1-S7 are injected into theirrespective vertical flow channels 102. The reagents in each horizontalflow channel 104 thus mix with the samples in each of the vertical flowchannels 102 at the intersections 106, which in this particular deviceare in the shape of a well or chamber. Thus, in the specific case of anucleic acid amplification reaction, for example, the reagents necessaryfor the amplification reaction are introduced into each of thehorizontal flow channels 104. Different nucleic acid templates areintroduced into the vertical flow channels 102. In certain analyses, theprimer introduced as part of the reagent mixture that is introduced intoeach of the horizontal flow channels 104 might differ between flowchannels. This allows each nucleic acid template to be reacted with anumber of different primers.

FIGS. 1B-E show enlarged plan views of adjacent reaction sites in thedevice depicted in FIG. 1A to illustrate more specifically how thedevice operates during an analysis. For the purposes of clarity, theintersections 106 are not shown in the form of reaction wells andcontrol channels 116, 118 have been omitted, with just the column androw valves 110, 108 being shown (rectangular boxes). As shown in FIG.1B, an analysis is commenced by closing column valves 110 and openingrow valves 108 to allow solution flow through horizontal flow channel104 while blocking flow through vertical flow channels 102. Reagent R1is then introduced into horizontal flow channel 104 and flowedcompletely through the length of the horizontal flow channel 104 suchthat all the reaction sites 106 are filled. Solution flow throughhorizontal channel 104 can be achieved by an external pump, but moretypically is achieved by incorporating a peristaltic pump into theelastomeric device itself as described in detail in Unger et al. (2000)Science 288:113-116, and PCT Publication WO 01/01025, for example.

Once R1 has been introduced, row valves 108 are closed and column valves102 opened (see FIG. 1C). This allows samples S1 and S2 to be introducedinto vertical flow channels 102 and to flow through their respectiveflow channels. As the samples flow through the vertical flow channels102, they expel R1 from the reaction sites 106, thus leaving sample atreaction sites 106. Then, as shown in FIG. 1D, row valves 108 are openedto allow S1 and S2 to diffuse and mix with R1. Thus, a mixture of sampleand reactant (R1S1 and R1S2) is obtained in the region of eachintersection or reaction site 106. After allowing a sufficient time forS1 and S2 to diffuse with R1, all row and column valves 108, 110 areclosed to isolate S1 and S2 within the region of their respectivereaction sites 106 and to prevent intermixing of S1 and S2 (see FIG.1E). The mixtures are then allowed to react and the reactions detectedby monitoring the intersection 106 or the cross-shaped region thatincludes the intersection 106. For analyses requiring heating (e.g.,thermocycling during amplification reactions), the device is placed on aheater and heated while the samples remain isolated.

A modified version of the device shown in FIG. 1A is shown in FIG. 1F.The general structure bears many similarities with that depicted in FIG.1A, and common elements in both figures share the same referencenumbers. The device 150 illustrated in FIG. 1F differs in that pairs ofhorizontal flow channels 104 are joined to a common inlet 124. Thisessentially enables duplicate sets of reagents to be introduced into twoadjacent flow channels with just a single injection into inlet 124. Theuse of a common inlet is further extended with respect to the verticalflow channels 102. In this particular example, each sample is introducedinto five vertical flow channels 102 with a single injection into sampleinlet 120. Thus, with this particular device, there are essentially tenreplicate reactions for each particular combination of sample andreagent. Of course, the number of replicate reactions can be varied asdesired by altering the number of vertical and/or horizontal flowchannels 102, 104 that are joined to a common inlet 120, 124.

The device shown in FIG. 1F also includes a separate control channelinlet 128 that regulates control channel 130 that can be used to governsolution flow toward outlets 132 and another control channel inlet 132that regulates control channel 134 that regulates solution flow tooutlets 136. Additionally, device 150 incorporates guard channels 138.In this particular design, the guard channels 138 are formed as part ofcontrol channels 116. As indicated supra, the guard channels 138 aresmaller than the row valves 108; consequently, the membranes of theguard channels 138 are not deflected into the underlying horizontal flowchannels 104 such that solution flow is disrupted.

Finally, the design shown in FIG. 1F differs in that reaction does notoccur in wells at the intersection of the horizontal and vertical flowlines, but in the intersection itself.

V. Blind Channel Designs

A. General

Devices utilizing a blind channel design have certain features. First,the devices include one or more flow channels from which one or moreblind channels branch. As indicated above, the end region of suchchannels can serve as a reaction site. A valve formed by an overlayingflow channel can be actuated to isolate the reaction site at the end ofthe blind channel. The valves provide a mechanism for switchablyisolating the reaction sites.

Second, the flow channel network in communication with the blindchannels is configured such that all or most of the reaction sites canbe filled with a single or a limited number of inlets (e.g., less than 5or less than 10). The ability to fill a blind flow channel is madepossible because the devices are made from elastomeric material. Theelastomeric material is sufficiently porous such that air within theflow channels and blind channels can escape through these pores assolution is introduced into the channels. The lack of porosity ofmaterials utilized in other microfluidic devices precludes use of theblind channel design because air in a blind channel has no way to escapeas solution is injected.

A third characteristic is that one or more reagents are non-covalentlydeposited on a base layer of elastomer during manufacture (see infra forfurther details on the fabrication process) within the reaction sites.The reagent(s) are non-covalently attached because the reagents aredesigned to become dissolved when sample is introduced into the reactionsite. To maximize the number of analyses, a different reactant or set ofreactants is deposited at each of the different reaction sites.

Certain blind channel devices are designed such that the reaction sitesare arranged in the form of an array.

Thus, in those blind channel devices designed for conducting nucleicacid amplification reactions, for example, one or more of the reagentsrequired for conducting the extension reaction are deposited at each ofthe reaction sites during manufacture of the device. Such reagentsinclude, for example, all or some of the following: primers, polymerase,nucleotides, cofactors, metal ions, buffers, intercalating dyes and thelike. To maximize high throughput analysis, different primers selectedto amplify different regions of DNA are deposited at each reaction site.Consequently, when a nucleic acid template is introduced into thereaction sites via inlet, a large number of extension reactions can beperformed at different segments of the template. Thermocycling necessaryfor an amplification reaction can be accomplished by placing the deviceon a thermocycling plate and cycling the device between the variousrequired temperatures.

The reagents can be immobilized in a variety of ways. For example, insome instances one or more of the reagents are non-covalently depositedat the reaction site, whereas in other instances one or more of thereagents is covalently attached to the substrate at the reaction site.If covalently attached, the reagents can be linked to the substrate viaa linker. A variety of linker types can be utilized such asphotochemical/photolabile linkers, themolabile linkers, and linkers thatcan be cleaved enzymatically. Some linkers are bifunctional (i.e., thelinker contains a functional group at each end that is reactive withgroups located on the element to which the linker is to be attached);the functional groups at each end can be the same or different. Examplesof suitable linkers that can be used in some assays include straight orbranched-chain carbon linkers, heterocyclic linkers and peptide linkers.A variety of types of linkers are available from Pierce Chemical Companyin Rockford, Ill. and are described in EPA 188,256; U.S. Pat. Nos.4,671,958; 4,659,839; 4,414,148; 4,669,784; 4,680,338, 4,569,789 and4,589,071, and by Eggenweiler, H. M, Pharmaceutical Agent DiscoveryToday 1998, 3, 552. NVOC (6 nitroveratryloxycarbonyl) linkers and otherNVOC-related linkers are examples of suitable photochemical linkers(see, e.g., WO 90/15070 and WO 92/10092). Peptides that have proteasecleavage sites are discussed, for example, in U.S. Pat. No. 5,382,513.

B. Exemplary Designs and Uses

FIG. 2 is a simplified plan view of one exemplary device utilizing theblind channel design. The device 200 includes a flow channel 204 and aset of branch flow channels 206 branching therefrom that are formed inan elastomeric substrate 202. Each branch flow channel 206 terminates ina reaction site 208, thereby forming an array of reaction sites.Overlaying the branch flow channels 206 is a control channel 210 that isseparated from the branch flow channels 206 by membranes 212. Actuationof control channel 210 causes membranes 212 to deflect into the branchflow channels 206 (i.e., to function as a valve), thus enabling each ofthe reaction sites 208 to be isolated from the other reaction sites.

Operation of such a device involves injecting a test sample into flowchannel 204 with solution subsequently flowing into each of branchchannels 206. Once the sample has filled each branch channel 206,control channel 210 is actuated to cause activation of valves/membranes212 to deflect into branch channels 206, thereby sealing off each ofreaction sites 208. As the sample flows into and remains in reactionsites 208, it dissolves reagents previously spotted at each of thereaction sites 208. Once dissolved, the reagents can react with thesample. Valves 212 prevent the dissolved reagents at each reaction site208 from intermixing by diffusion. Reaction between sample and reagentsare then detected, typically within reaction site 208. Reactions canoptionally be heated as described in the temperature control sectioninfra.

FIG. 3A illustrates an example of a somewhat more complex blind flowchannel design. In this particular design 300, each of a set ofhorizontal flow channels 304 are connected at their ends to two verticalflow channels 302. A plurality of branch flow channels 306 extend fromeach of the horizontal flow channels 304. The branch flow channels 304in this particular design are interleaved such that the branch channel306 attached to any given horizontal flow channel 304 is positionedbetween two branch channels 306 joined to an immediately adjacenthorizontal flow channel 304, or positioned between a branch flow channel306 joined to an immediately adjacent flow channel 304 and one of thevertical flow channels 302. As with the design depicted in FIG. 3A, eachbranch flow channel 306 terminates in a reaction site 308. Alsoconsistent with the design shown in FIG. 3A, a control channel 310overlays each of the branch channels and is separated from theunderlying branch channel by membrane 312. The control channel isactuated at port 316. The vertical and horizontal flow channels 302, 304are interconnected such that injection of sample into inlet 314 allowssolution to flow throughout the horizontal and vertical flow channelnetwork and ultimately into each of the reaction sites 308 via thebranch flow channels 306.

Hence, in operation, sample is injected into inlet to introduce solutioninto each of the reaction sites. Once the reaction sites are filled,valves/membranes are actuated to trap solution within the reaction sitesby pressurizing the control channels at port. Reagents previouslydeposited in the reaction sites become resuspended within the reactionsites, thereby allowing reaction between the deposited reagents andsample within each reaction site. Reactions within the reaction sitesare monitored by a detector. Again, reactions can optionally becontrollably heated according to the methods set forth in thetemperature control section below.

An even more complicated version of the general design illustrated inFIG. 3A is shown in FIG. 3B. The device shown in FIG. 3B is one in whichthe unit organization of the horizontal and branch flow channels 302shown in FIG. 3A is repeated multiple times. The device shown in FIG. 3Bfurther illustrates the inclusion of guard channels 320 in those devicesto be utilized in applications that involve heating (e.g.,thermocycling). An exemplary orientation of the guard channels 320 withrespect to the flow channels 304 and branch channels 306 is shown in theenlarged view depicted in the right side of the drawing. The guardchannels 320 overlay the branch flow channels 306 and reaction sites308. As discussed above, water is flowed through the guard channels 320during heating of the device 300 to increase the local concentration ofwater in the device, thereby reducing evaporation of water from solutionin the flow channels 306 and reaction sites 308.

The features of blind channel devices discussed at the outset of thissection minimizes the footprint of the device and enable a large numberof reaction sites to be formed on the device and for high densities tobe obtained. For example, devices of this type having 2500 reactionsites can readily be manufactured to fit on a standard microscope slides(25 mm×75 mm). The foregoing features also enable very high densities ofreaction sites to be obtained with devices utilizing the blind channeldesign. For example, densities of at least 50, 60, 70, 80, 90 or 100reaction sites/cm² or any integral density value therebetween can bereadily obtained. However, certain devices have even higher densitiesranging, for example, between 100 to 4000 reaction sites/cm², or anyintegral density value therebetween. For instance, some devices havedensities of at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900 or 1000 sites/cm². Devices with very high densities of at least,2000, 3000, or 4000 sites/cm² are also obtainable. Such high densitiesdirectly translate into a very large number of reaction sites on thedevice. Devices utilizing the blind channel architecture typically haveat least 10-100 reaction sites, or any integral number of sitestherebetween. More typically, the devices have at least 100-1,000reaction sites, or any integral number of sites therebetween. Higherdensity devices can have even more reaction sites, such as at least1,000-100,000 reaction sites, or any integral number of sitestherebetween. Thus, certain devices have at least 100; 500; 1,000;2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; 20,000;30,000; 40,000; 50,000; or 100,000 reaction sites depending upon theoverall size of the device.

The large number of reaction sites and densities that can be obtained isalso a consequence of the ability to fabricate very small wells orcavities. For example, the cavities or wells typically have a volume ofless than 50 mL; in other instances less than 40 mL, 30 mL, 20 mL or 10mL; and in still other instances less than 5 mL or 1 mL. As a specificexample, certain devices have wells that are 300 microns long, 300microns wide and 10 microns deep.

The blind channel devices provided herein can utilize certain designfeatures and methodologies discussed in PCT Applications PCT/US01/44549(published as WO 02/43615) and PCT/US02/10875 (published as WO02/082047), including, for example, strategies for filling dead-endedchannels, liquid priming, pressurized outgas priming, as well as variousstrategies for displacing gas during the filling of microfluidicchannels. Both of these PCT publications are incorporated herein byreference in their entirety for all purposes.

VI. Hybrid Designs

Still other devices are hybrids of the matrix and blind fill designs.The design of devices of this type is similar to the blind channeldevice shown in FIG. 3A, except that each horizontal flow channel isconnected to its own sample inlet port(s) and the horizontal flowchannels are not interconnected via vertical flow channels.Consequently, sample introduced into any given horizontal flow channelfills only that horizontal flow channel and reaction sites attachedthereto. Whereas, in the blind flow channel device shown in FIG. 3A,sample can flow between the horizontal flow channels 304 via verticalflow channels 302.

An example of devices of this general device is shown in FIG. 4. Device400 comprises a plurality of horizontal flow channels 404, each of whichhas a plurality of branch flow channels 406 extending from it and itsown sample inlet 414. A control channel 410 overlays each of the branchflow channels 406 and membrane (valve) 412 separates the control channel410 from the underlying branch flow channel 406. As with the blind flowchannel design, actuation of the control channel at inlet 416 causesdeflection of membranes 412 into the branch flow channels 406 andisolation of reaction sites 408. In a variation of this design, eachhorizontal flow channel 404 can include an inlet 414 at each end, thusallowing sample to be introduced from both ends.

In some instances, reagents are deposited at the reaction sites duringmanufacture of the device. This enables a large number of samples to betested under a relatively large number of reaction conditions in a shortperiod of time without requiring time-consuming additions of reagents asrequired with the matrix devices. Alternatively, reaction mixtures canbe prepared prior to injection on the chip. Once the mixtures areinjected, they can be analyzed or further treated (e.g., heated).

By injecting different samples into each of the horizontal flowchannels, a large number of samples can be rapidly analyzed. Assumingreagents have been previously deposited at the reaction sites, thepresence of the same reagent at each reaction site associated with anygiven horizontal flow channel provides a facile way to conduct a numberof replicate reactions with each sample. If instead, the reagent at thereaction sites differ for any given flow channel, then each sample isessentially simultaneously exposed to a variety of different reactionconditions.

Thus, the devices provided herein are tailored for a variety ofdifferent types of investigations. If an investigation involvesscreening of a relatively large number of different samples under usercontrolled conditions (e.g., 100 samples against 100 user selectedreagents), then the matrix devices provide a useful solution. If,however, the investigation involves analyzing one or a limited number ofsamples under a wide variety of reaction conditions (e.g., one sampleagainst 10,000 reaction conditions), then the blind channel design isuseful. Finally, if one wants to examine a relatively large number ofsamples against defined reaction conditions without having to injectreagents (e.g., 100 samples against 100 previously defined reagents),then the hybrid devices are useful.

VII. Temperature Control

A. Devices and Components

A number of different options of varying sophistication are availablefor controlling temperature within selected regions of the microfluidicdevice or the entire device. Thus, as used herein, the term temperaturecontroller is meant broadly to refer to a device or element that canregulate temperature of the entire microfluidic device or within aportion of the microfluidic device (e.g., within a particulartemperature region or at one or more junctions in a matrix of blindchannel-type microfluidic device).

Generally, the devices are placed on a thermal cycling plate to thermalcycle the device. A variety of such plates are readily available fromcommercial sources, including for example the ThermoHybaid Px2(Franklin, Mass.), MJ Research PTC-200 (South San Francisco, Calif.),Eppendorf Part# E5331 (Westbury, N.Y.), Techne Part #205330 (Princeton,N.J.).

To ensure the accuracy of thermal cycling steps, in certain devices itis useful to incorporate sensors detecting temperature at variousregions of the device. One structure for detecting temperature is athermocouple. Such a thermocouple could be created as thin film wirespatterned on the underlying substrate material, or as wires incorporateddirectly into the microfabricated elastomer material itself.

Temperature can also be sensed through a change in electricalresistance. For example, change in resistance of a thermistor fabricatedon an underlying semiconductor substrate utilizing conventionaltechniques can be calibrated to a given temperature change.Alternatively, a thermistor could be inserted directly into themicrofabricated elastomer material. Still another approach to detectionof temperature by resistance is described in Wu et al. in “MEMS FlowSensors for Nano-fluidic Applications”, Sensors and Actuators A 89152-158 (2001), which is hereby incorporated by reference in itsentirety. This paper describes the use of doped polysilicon structuresto both control and sense temperature. For polysilicon and othersemiconductor materials, the temperature coefficient of resistance canbe precisely controlled by the identity and amount of dopant, therebyoptimizing performance of the sensor for a given application.

Thermo-chromatic materials are another type of structure available todetect temperature on regions of an amplification device. Specifically,certain materials dramatically and reproducibly change color as theypass through different temperatures. Such a material could be added tothe solution as they pass through different temperatures.Thermo-chromatic materials could be formed on the underlying substrateor incorporated within the elastomer material. Alternatively,thermo-chromatic materials could be added to the sample solution in theform of particles.

Another approach to detecting temperature is through the use of aninfrared camera. An infrared camera in conjunction with a microscopecould be utilized to determine the temperature profile of the entireamplification structure. Permeability of the elastomer material toradiation would facilitate this analysis.

Yet another approach to temperature detection is through the use ofpyroelectric sensors. Specifically, some crystalline materials,particularly those materials also exhibiting piezoelectric behavior,exhibit the pyroelectric effect. This effect describes the phenomena bywhich the polarization of the material's crystal lattice, and hence thevoltage across the material, is highly dependent upon temperature. Suchmaterials could be incorporated onto the substrate or elastomer andutilized to detect temperature.

Other electrical phenomena, such as capacitance and inductance, can beexploited to detect temperature in accordance with embodiments of thepresent invention.

B. Verification of Accurate Thermocycling

As described in greater detail in the fabrication section infra, blindchannel devices have a base layer onto which reagents are placed. Thestructure comprising the two layers containing the flow channels andcontrol channels is overlayed on the base layer such that the flowchannels are aligned with the deposited reagents. The other side of thebase layer is then placed upon a substrate (e.g., glass). Usually, thereaction site at which reaction occurs is about 100-150 microns abovethe substrate/glass interface. Using known equations for thermaldiffusivity and appropriate values for the elastomers and glass utilizedin the device, one can calculate the time required for the temperaturewithin the reaction site to reach the temperature the controller seeksto maintain. The calculated values shown in Table 1 demonstrate thattemperature can rapidly be reached, even using elastomer and glasslayers considerably thicker than utilized in devices in which thereaction site is approximately 100-150 microns (i.e., the typicaldistance for the devices described herein).

TABLE 1 Calculated heat diffusion lengths through PDMS and glass layersat the indicated time periods. 1 second 10 seconds 100 seconds PDMS 400um 1.26 mm 4.0 mm Glass 640 um  2.0 mm 6.4 mm

FIG. 5 illustrates the rapidity at which the desired temperature isachieved using a blind channel device.

In another embodiment, temperature may be measured by using doublestranded oligonucleotide polymers having known tms wherein anintercollating dye whose intercollation indicates the whether theoligonucleotide is hybridized or denatured, such as SYBR Green™ orethidium bromide for example, wherein by introducing a solutioncontaining the oligonucleotide with the dye into the chambers of themicrofluidic device having an array of reaction chambers can be used todetermine the extent to which the temperature of each chamber isconsistent across the array. In this embodiment, as the temperature israised above the tm, the intercolating dye changes its relation to theoligonucleotide upon it sdenataturation into a single strandedoligonucleotide. Alternatively, the if the temperature is above the tmand is lowered, an the intercollation of the dye into the now annealedoligonucleotide may be monitored. The use of the dye in essence providesfor an “oligonucleotide thermometer” which changes a property, such asfluorescence, in response to a temperature change relative to the tm ofthe oligonucleotide. By designing or using oligonucleotides of aselected tm, the extent to which an array of reaction chambers changetemperature in a similar manner can be determined.

VIII. Detection

A. General

A number of different detection strategies can be utilized with themicrofluidic devices that are provided herein. Selection of theappropriate system is informed in part on the type of event and/or agentbeing detected. The detectors can be designed to detect a number ofdifferent signal types including, but not limited to, signals fromradioisotopes, fluorophores, chromophores, electron dense particles,magnetic particles, spin labels, molecules that emit chemiluminescence,electrochemically active molecules, enzymes, cofactors, enzymes linkedto nucleic acid probes and enzyme substrates.

Illustrative detection methodologies suitable for use with the presentmicrofluidic devices include, but are not limited to, light scattering,multichannel fluorescence detection, UV and visible wavelengthabsorption, luminescence, differential reflectivity, and confocal laserscanning. Additional detection methods that can be used in certainapplication include scintillation proximity assay techniques,radiochemical detection, fluorescence polarization, fluorescencecorrelation spectroscopy (FCS), time-resolved energy transfer (TRET),fluorescence resonance energy transfer (FRET) and variations such asbioluminescence resonance energy transfer (BRET). Additional detectionoptions include electrical resistance, resistivity, impedance, andvoltage sensing.

Detection occurs at a “detection section,” or “detection region.” Theseterms and other related terms refer to the portion of the microfluidicdevice at which detection occurs. As indicated above, with devicesutilizing the blind channel design, the detection section is generallythe reaction site as isolated by the valve associated with each reactionsite. The detection section for matrix-based devices is usually withinregions of flow channels that are adjacent an intersection, theintersection itself, or a region that encompasses the intersection and asurrounding region.

The detection section can be in communication with one or moremicroscopes, diodes, light stimulating devices (e.g., lasers),photomultiplier tubes, processors and combinations of the foregoing,which cooperate to detect a signal associated with a particular eventand/or agent. Often the signal being detected is an optical signal thatis detected in the detection section by an optical detector. The opticaldetector can include one or more photodiodes (e.g., avalanchephotodiodes), a fiber-optic light guide leading, for example, to aphotomultiplier tube, a microscope, and/or a video camera (e.g., a CCDcamera).

Detectors can be microfabricated within the microfluidic device, or canbe a separate element. If the detector exists as a separate element andthe microfluidic device includes a plurality of detection sections,detection can occur within a single detection section at any givenmoment. Alternatively, scanning systems can be used. For instance,certain automated systems scan the light source relative to themicrofluidic device; other systems scan the emitted light over adetector, or include a multichannel detector. As a specific illustrativeexample, the microfluidic device can be attached to a translatable stageand scanned under a microscope objective. A signal so acquired is thenrouted to a processor for signal interpretation and processing. Arraysof photomultiplier tubes can also be utilized. Additionally, opticalsystems that have the capability of collecting signals from all thedifferent detection sections simultaneously while determining the signalfrom each section can be utilized.

External detectors are usable because the devices that are provided arecompletely or largely manufactured of materials that are opticallytransparent at the wavelength being monitored. This feature enables thedevices described herein to utilize a number of optical detectionsystems that are not possible with conventional silicon-basedmicrofluidic devices.

A particularly preferred detector uses a CCD camera and an optical paththat provides for a large field of view and a high numerical aperture tomaximize the amount of light collected from each reaction chamber. Inthis regard, the CCD is used as an array of photodetectors wherein eachpixel or group of pixels corresponds to a reaction chamber rather thanbeing used to produce an image of the array. Thus, the optics may bealtered such that image quality is reduced or defocused to increase thedepth of field of the optical system to collect more light from eachreaction chamber.

A detector can include a light source for stimulating a reporter thatgenerates a detectable signal. The type of light source utilized dependsin part on the nature of the reporter being activated. Suitable lightsources include, but are not limited to, lasers, laser diodes and highintensity lamps. If a laser is utilized, the laser can be utilized toscan across a set of detection sections or a single detection section.Laser diodes can be microfabricated into the microfluidic device itself.Alternatively, laser diodes can be fabricated into another device thatis placed adjacent to the microfluidic device being utilized to conducta thermal cycling reaction such that the laser light from the diode isdirected into the detection section.

Detection can involve a number of non-optical approaches as well. Forexample, the detector can also include, for example, a temperaturesensor, a conductivity sensor, a potentiometric sensor (e.g., pHelectrode) and/or an amperometric sensor (e.g., to monitor oxidation andreduction reactions).

A number of commercially-available external detectors can be utilized.Many of these are fluorescent detectors because of the ease in preparingfluorescently labeled reagents. Specific examples of detectors that areavailable include, but are not limited to, Applied Precision ArrayWoRx(Applied Precision, Issaquah, Wash.)).

B. Detection of Amplified Nucleic Acids

1. Intercalation Dyes

Certain intercalation dyes that only fluoresce upon binding todouble-stranded DNA can be used to detect double-stranded amplified DNA.Examples of suitable dyes include, but are not limited to, SYBR™ andPico Green (from Molecular Probes, Inc. of Eugene, Oreg.), ethidiumbromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258,Toto-1, Yoyo-1, and DAPI (4′,6-diamidino-2-phenylindole hydrochloride).Additional discussion regarding the use of intercalation dyes isprovided by Zhu et al., Anal. Chem. 66:1941-1948 (1994), which isincorporated by reference in its entirety.

2. FRET Based Detection Methods

Detection methods of this type involve detecting a change influorescence from a donor (reporter) and/or acceptor (quencher)fluorophore in a donor/acceptor fluorophore pair. The donor and acceptorfluorophore pair are selected such that the emission spectrum of thedonor overlaps the excitation spectrum of the acceptor. Thus, when thepair of fluorophores are brought within sufficiently close proximity toone another, energy transfer from the donor to the acceptor can occur.This energy transfer can be detected.

FRET and Template Extension Reactions.

These methods generally utilize a primer labeled with one member of adonor/acceptor pair and a nucleotide labeled with the other member ofthe donor/acceptor pair. Prior to incorporation of the labelednucleotide into the primer during an template-dependent extensionreaction, the donor and acceptor are spaced far enough apart that energytransfer cannot occur. However, if the labeled nucleotide isincorporated into the primer and the spacing is sufficiently close, thenenergy transfer occurs and can be detected. These methods areparticularly useful in conducting single base pair extension reactionsin the detection of single nucleotide polymorphisms (see infra) and aredescribed in U.S. Pat. No. 5,945,283 and PCT Publication WO 97/22719.

Quantitative RT-PCR.

A variety of so-called “real time amplification” methods or “real timequantitative PCR” methods can also be utilized to determine the quantityof a target nucleic acid present in a sample by measuring the amount ofamplification product formed during or after the amplification processitself. Fluorogenic nuclease assays are one specific example of a realtime quantitation method which can be used successfully with the devicesdescribed herein. This method of monitoring the formation ofamplification product involves the continuous measurement of PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature as the “TaqMan”method.

The probe used in such assays is typically a short (ca. 20-25 bases)polynucleotide that is labeled with two different fluorescent dyes. The5′ terminus of the probe is typically attached to a reporter dye and the3′ terminus is attached to a quenching dye, although the dyes can beattached at other locations on the probe as well. The probe is designedto have at least substantial sequence complementarity with the probebinding site on the target nucleic acid. Upstream and downstream PCRprimers that bind to regions that flank the probe binding site are alsoincluded in the reaction mixture.

When the probe is intact, energy transfer between the two fluorophorsoccurs and the quencher quenches emission from the reporter. During theextension phase of PCR, the probe is cleaved by the 5′ nuclease activityof a nucleic acid polymerase such as Taq polymerase, thereby releasingthe reporter from the polynucleotide-quencher and resulting in anincrease of reporter emission intensity which can be measured by anappropriate detector.

One detector which is specifically adapted for measuring fluorescenceemissions such as those created during a fluorogenic assay is the ABI7700 manufactured by Applied Biosystems, Inc. in Foster City, Calif.Computer software provided with the instrument is capable of recordingthe fluorescence intensity of reporter and quencher over the course ofthe amplification. These recorded values can then be used to calculatethe increase in normalized reporter emission intensity on a continuousbasis and ultimately quantify the amount of the mRNA being amplified.

Additional details regarding the theory and operation of fluorogenicmethods for making real time determinations of the concentration ofamplification products are described, for example, in U.S. Pat. Nos.5,210,015 to Gelfand, 5,538,848 to Livak, et al., and 5,863,736 toHaaland, as well as Heid, C. A., et al., Genome Research, 6:986-994(1996); Gibson, U. E. M, et al., Genome Research 6:995-1001 (1996);Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991);and Livak, K. J., et al., PCR Methods and Applications 357-362 (1995),each of which is incorporated by reference in its entirety.

Thus, as the amplification reaction progresses, an increasing amount ofdye becomes bound and is accompanied by a concomitant increase insignal.

Intercalation dyes such as described above can also be utilized in adifferent approach to quantitative PCR methods. As noted above, thesedyes preferentially bind to double stranded DNA (e.g., SYBR GREEN) andonly generate signal once bound. Thus, as an amplification reactionprogresses, an increasing amount of dye becomes bound and is accompaniedby a concomitant increase in signal that can be detected.

Molecular Beacons:

With molecular beacons, a change in conformation of the probe as ithybridizes to a complementary region of the amplified product results inthe formation of a detectable signal. The probe itself includes twosections: one section at the 5′ end and the other section at the 3′ end.These sections flank the section of the probe that anneals to the probebinding site and are complementary to one another. One end section istypically attached to a reporter dye and the other end section isusually attached to a quencher dye.

In solution, the two end sections can hybridize with each other to forma hairpin loop. In this conformation, the reporter and quencher dye arein sufficiently close proximity that fluorescence from the reporter dyeis effectively quenched by the quencher dye. Hybridized probe, incontrast, results in a linearized conformation in which the extent ofquenching is decreased. Thus, by monitoring emission changes for the twodyes, it is possible to indirectly monitor the formation ofamplification product. Probes of this type and methods of their use isdescribed further, for example, by Piatek, A. S., et al., Nat.Biotechnol. 16:359-63 (1998); Tyagi, S. and Kramer, F. R., NatureBiotechnology 14:303-308 (1996); and Tyagi, S. et al., Nat. Biotechnol.16:49-53 (1998), each of which is incorporated by reference herein intheir entirety for all purposes.

Invader:

Invader assays (Third Wave Technologies, (Madison, Wis.)) are used forSNP genotyping and utilize an oligonucleotide, designated the signalprobe, that is complementary to the target nucleic acid (DNA or RNA) orpolymorphism site. A second oligonucleotide, designated the InvaderOligo, contains the same 5′ nucleotide sequence, but the 3′ nucleotidesequence contains a nucleotide polymorphism. The Invader Oligointerferes with the binding of the signal probe to the target nucleicacid such that the 5′ end of the signal probe forms a “flap” at thenucleotide containing the polymorphism. This complex is recognized by astructure specific endonuclease, called the Cleavase enzyme. Cleavasecleaves the 5′ flap of the nucleotides. The released flap binds with athird probe bearing FRET labels, thereby forming another duplexstructure recognized by the Cleavase enzyme. This time the Cleavaseenzyme cleaves a fluorophore away from a quencher and produces afluorescent signal. For SNP genotyping, the signal probe will bedesigned to hybridize with either the reference (wild type) allele orthe variant (mutant) allele. Unlike PCR, there is a linear amplificationof signal with no amplification of the nucleic acid. Further detailssufficient to guide one of ordinary skill in the art is provided by, forexample, Neri, B. P., et al., Advances in Nucleic Acid and ProteinAnalysis 3826:117-125, 2000).

Nasba:

Nucleic Acid Sequence Based Amplification (NASBA) is a detection methodusing RNA as the template. A primer complementary to the RNA containsthe sequence for the T7 promoter site. This primer is allowed to bindwith the template RNA and Reverse Transcriptase (RT) added to generatethe complementary strand from 3′ to 5′. RNase H is subsequently added todigest away the RNA, leaving single stranded cDNA behind. A second copyof the primer can then bind the single stranded cDNA and make doublestranded cDNA. T7 RNA polymerase is added to generate many copies of theRNA from the T7 promoter site that was incorporated into the cDNAsequence by the first primer. All the enzymes mentioned are capable offunctioning at 41° C. (See, e.g., Compton, J. Nucleic AcidSequence-based Amplification, Nature 350: 91-91, 1991.)

Scorpion. This method is described, for example, by Thelwell N., et al.Nucleic Acids Research, 28:3752-3761, 2000, which is hereby incorporatedby reference in its entirety for all purposes, and which FIG. 20 depictsthe scheme thereof, wherein Scorpion probing mechanism is as follows.Step 1: initial denaturation of target and Scorpion stem sequence. Step2: annealing of Scorpion primer to target. Step 3: extension of Scorpionprimer produces double-stranded DNA. Step 4: denaturation ofdouble-stranded DNA produced in step 3. This gives a single-strandedtarget molecule with the Scorpion primer attached. Step 5: on cooling,the Scorpion probe sequence binds to its target in an intramolecularmanner. This is favoured over the intermolecular binding of thecomplementary target strand. A Scorpion (as shown in FIG. 20) consistsof a specific probe sequence that is held in a hairpin loopconfiguration by complementary stem sequences on the 5′ and 3′ sides ofthe probe. The fluorophore attached to the 5′-end is quenched by amoiety (normally methyl red) joined to the 3′-end of the loop. Thehairpin loop is linked to the 5′-end of a primer via a PCR stoppingsequence (stopper). After extension of the primer during PCRamplification, the specific probe sequence is able to bind to itscomplement within the same strand of DNA. This hybridization event opensthe hairpin loop so that fluorescence is no longer quenched and anincrease in signal is observed. The PCR stopping sequence preventsread-through, that could lead to opening of the hairpin loop in theabsence of the specific target sequence. Such read-through would lead tothe detection of non-specific PCR products, e.g. primer dimers ormispriming events.

3. Capacitive DNA Detection

There is a linear relationship between DNA concentration and the changein capacitance that is evoked by the passage of nucleic acids across a1-kHz electric field. This relationship has been found to be speciesindependent. (See, e.g., Sohn, et al. (2000) Proc. Natl. Acad. Sci.U.S.A. 97:10687-10690). Thus, in certain devices, nucleic acids withinthe flow channel (e.g., the substantially circular flow channel of FIG.1 or the reaction chambers of FIG. 2) are subjected to such a field todetermine concentration of amplified product. Alternatively, solutioncontaining amplified product is withdrawn and then subjected to theelectric field.

IX. Composition of Mixtures for Conducting Reactions

Reactions conducted with the microfluidic devices disclosed herein aretypically conducted with certain additives to enhance the reactions. So,for example, in the case of devices in which reagents are deposited,these additives can be spotted with one or more reactants at a reactionsite, for instance. One set of additives are blocking reagents thatblock protein binding sites on the elastomeric substrate. A wide varietyof such compounds can be utilized including a number of differentproteins (e.g., gelatin and various albumin proteins, such as bovineserum albumin) and glycerol.

A detergent additive can also be useful. Any of a number of differentdetergents can be utilized. Examples include, but are not limited to SDSand the various Triton detergents.

In the specific case of nucleic acid amplification reactions, a numberof different types of additives can be included. One category areenhancers that promote the amplification reaction. Such additivesinclude, but are not limited to, reagents that reduce secondarystructure in the nucleic acid (e.g., betaine), and agents that reducemispriming events (e.g., tetramethylammonium chloride).

It has also been found in conducting certain amplification reactionsthat some polymerases give enhanced results. For example, while goodresults were obtained with AmpliTaq Gold polymerase (Applied Biosystems,Foster City, Calif.) from Thermus aquaticus, improved reactions were insome instances obtained using DyNAzyme polymerase from Finnzyme, Espoo,Finland. This polymerase is from the thermophilic bacterium, Thermusbrockianus. Other exemplary polymerases that can be utilized include,but are not limited to, rTH polymerase XL, which is a combination ofThermus thermophilus (Tth) and Thermococcus litoralis (Tli),hyperthermo-philic archaebacterium Pyrosoccus woesei (Pwo), and Tgo DNAPolymerase.

Further details regarding additives useful in conducting reactions withcertain of the devices disclosed herein, including nucleic acidamplification reactions, are provided in Example 1 infra.

X. Exemplary Applications

Because the microfluidic devices provided herein can be manufactured toinclude a large number of reaction sites, the devices are useful in awide variety of screening and analytical methods. In general, thedevices can be utilized to detect reactions between species that reactto form a detectable signal, or a product that upon interaction withanother species generates a detectable signal. In view of their use withvarious types of temperature control systems, the devices can also beutilized in a number of different types of analyses or reactionsrequiring temperature control.

A. Nucleic Acid Amplification Reactions

The devices disclosed herein can be utilized to conduct essentially anytype of nucleic acid amplification reaction. Thus, for example,amplification reactions can be linear amplifications, (amplificationswith a single primer), as well as exponential amplifications (i.e.,amplifications conducted with a forward and reverse primer set).

When the blind channel type devices are utilized to perform nucleic acidamplification reactions, the reagents that are typically depositedwithin the reaction sites are those reagents necessary to perform thedesired type of amplification reaction. Usually this means that some orall of the following are deposited, primers, polymerase, nucleotides,metal ions, buffer, and cofactors, for example. The sample introducedinto the reaction site in such cases is the nucleic acid template.Alternatively, however, the template can be deposited and theamplification reagents flowed into the reaction sites. As discussedsupra, when the matrix device is utilized to conduct an amplificationreaction, samples containing nucleic acid template are flowed throughthe vertical flow channels and the amplification reagents through thehorizontal flow channels or vice versa.

While PCR is perhaps the best known amplification technique. The devicesare not limited to conducting PCR amplifications. Other types ofamplification reactions that can be conducted include, but are notlimited to, (i) ligase chain reaction (LCR) (see Wu and Wallace,Genomics 4:560 (1989) and Landegren et al., Science 241:1077 (1988));(ii) transcription amplification (see Kwoh et al., Proc. Natl. Acad.Sci. USA 86:1173 (1989)); (iii) self-sustained sequence replication (seeGuatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)); and (iv)nucleic acid based sequence amplification (NASBA) (see, Sooknanan, R.and Malek, L., BioTechnology 13: 563-65 (1995)). Each of the foregoingreferences are incorporated herein by reference in their entirety forall purposes.

Detection of the resulting amplified product can be accomplished usingany of the detection methods described supra for detecting amplifiedDNA.

B. SNP Analysis and Genotyping

1. General

Many diseases linked to genome modifications, either of the hostorganism or of infectious organisms, are the consequence of a change ina small number of nucleotides, frequently involving a change in a singlenucleotide. Such single nucleotide changes are referred to as singlenucleotide polymorphisms or simply SNPs, and the site at which the SNPoccurs is typically referred to as a polymorphic site. The devicesdescribed herein can be utilized to determine the identify of anucleotide present at such polymorphic sites. As an extension of thiscapability, the devices can be utilized in genotyping analyses.Genotyping involves the determination of whether a diploid organism(i.e., an organism with two copies of each gene) contains two copies ofa reference allele (a reference-type homozygote), one copy each of thereference and a variant allele (i.e., a heterozygote), or contains twocopies of the variant allele (i.e., a variant-type homozygote). Whenconducting a genotyping analysis, the methods of the invention can beutilized to interrogate a single variant site. However, as describedfurther below in the section on multiplexing, the methods can also beused to determine the genotype of an individual in many different DNAloci, either on the same gene, different genes or combinations thereof.

Devices to be utilized for conducting genotyping analyses are designedto utilize reaction sites of appropriate size to ensure from astatistical standpoint that a copy of each of the two alleles for adiploid subject are present in the reaction site at a workable DNAconcentrations. Otherwise, an analysis could yield results suggestingthat a heterozygote is a homozygote simply because a copy of the secondallele is not present at the reaction site. Table 2 below indicates thenumber of copies of the genome present in a 1 nl reaction volume atvarious exemplary DNA concentrations that can be utilized with thedevices described herein.

TABLE 2 Number of genome copies present in a 1 nL volume at theindicated DNA concentration. Volume (nL) [DNA] (ug/uL) N 1 0.33 100 10.10 32 1 0.05 16 1 0.01 3 1 0.003 1

As a general matter, due to stochastic proportioning of the sample, thecopy number present before an amplification reaction is commenceddetermines the likely error in the measurement. Genotyping analysesusing certain devices are typically conducted with samples having a DNAconcentration of approximately 0.10 ug/uL, although the currentinventors have run successful TaqMan reactions at concentrations inwhich there is a single genome per reaction site.

2. Methods

Genotyping analyses can be conducted using a variety of differentapproaches. In these methods, it is generally sufficient to obtain a“yes” or “no” result, i.e., detection need only be able to answer thequestion whether a given allele is present. Thus, analyses can beconducted only with the primers or nucleotides necessary to detect thepresence of one allele potentially at a polymorphic site. However, moretypically, primers and nucleotides to detect the presence of each allelepotentially at the polymorphic site are included. Examples of suitableapproaches follow.

Single Base Pair Extension (SBPE) Reactions.

SBPE reactions are one technique specifically developed for conductinggenotyping analyses. Although a number of SPBE assays have beendeveloped, the general approach is quite similar. Typically, theseassays involve hybridizing a primer that is complementary to a targetnucleic acid such that the 3′ end of the primer is immediately 5′ of thevariant site or is adjacent thereto. Extension is conducted in thepresence of one or more labeled non-extendible nucleotides that arecomplementary to the nucleotide(s) that occupy the variant site and apolymerase. The non-extendible nucleotide is a nucleotide analog thatprevents further extension by the polymerase once incorporated into theprimer. If the added non-extendible nucleotide(s) is(are) complementaryto the nucleotide at the variant site, then a labeled non-extendiblenucleotide is incorporated onto the 3′ end of the primer to generate alabeled extension product. Hence, extended primers provide an indicationof which nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657.

Detection of the extended products can be detected utilizing the FRETdetection approach described for extension reactions in the detectionsection supra. Thus, for example, using the devices described herein, areagent mixture containing a primer labeled with one member of adonor/acceptor fluorophore, one to four labeled non-extendiblenucleotides (differentially labeled if more than one non-extendiblenucleotide is included), and polymerase are introduced (or previouslyspotted) at a reaction site. A sample containing template DNA is thenintroduced into the reaction site to allow template extension to occur.Any extension product formed is detected by the formation of a FRETsignal (see, e.g., U.S. Pat. No. 5,945,283 and PCT Publication WO97/22719.). The reactions can optionally be thermocycled to increasesignal using the temperature control methods and apparatus describedabove.

Quantitative PCR.

Genotyping analyses can also be conducted using the quantitative PCRmethods described earlier. In this case, differentially labeled probescomplementary to each of the allelic forms are included as reagents,together with primers, nucleotides and polymerase. However, reactionscan be conducted with only a single probe, although this can createambiguity as to whether lack of signal is due to absence of a particularallele or simply a failed reaction. For the typical biallelic case inwhich two alleles are possible for a polymorphic site, twodifferentially labeled probes, each perfectly complementary to one ofthe alleles are usually included in the reagent mixture, together withamplification primers, nucleotides and polymerase. Sample containing thetarget DNA is introduced into the reaction site. If the allele to whicha probe is complementary is present in the target DNA, thenamplification occurs, thereby resulting in a detectable signal asdescribed in the detection above. Based upon which of the differentialsignal is obtained, the identity of the nucleotide at the polymorphicsite can be determined. If both signals are detected, then both allelesare present. Thermocycling during the reaction is performed as describedin the temperature control section supra.

B. Gene Expression Analysis

1. General

Gene expression analysis involves determining the level at which one ormore genes is expressed in a particular cell. The determination can bequalitative, but generally is quantitative. In a differential geneexpression analysis, the levels of the gene(s) in one cell (e.g., a testcell) are compared to the expression levels of the same genes in anothercell (control cell). A wide variety of such comparisons can be made.Examples include, but are not limited to, a comparison between healthyand diseased cells, between cells from an individual treated with onedrug and cells from another untreated individual, between cells exposedto a particular toxicant and cells not exposed, and so on. Genes whoseexpression levels vary between the test and control cells can serve asmarkers and/or targets for therapy. For example, if a certain group ofgenes is found to be up-regulated in diseased cells rather than healthycells, such genes can serve as markers of the disease and canpotentially be utilized as the basis for diagnostic tests. These genescould also be targets. A strategy for treating the disease might includeprocedures that result in a reduction of expression of the up-regulatedgenes.

The design of the devices disclosed herein is helpful in facilitating avariety of gene expression analyses. Because the devices contain a largenumber of reaction sites, a large number of genes and/or samples can betested at the same time. Using the blind flow channel devices, forinstance, the expression levels of hundreds or thousands of genes can bedetermined at the same time. The devices also facilitate differentialgene expression analyses. With the matrix design, for example, a sampleobtained from a healthy cell can be tested in one flow channel, with asample from a diseased cell run in an immediately adjacent channel. Thisfeature enhances the ease of detection and the accuracy of the resultsbecause the two samples are run on the same device at the same time andunder the same conditions.

2. Sample Preparation and Concentration

To measure the transcription level (and thereby the expression level) ofa gene or genes, a nucleic acid sample comprising mRNA transcript(s) ofthe gene(s) or gene fragments, or nucleic acids derived from the mRNAtranscript(s) is obtained. A nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, are all derived from the mRNA transcript and detection ofsuch derived products is indicative of the presence and/or abundance ofthe original transcript in a sample. Thus, suitable samples include, butare not limited to, mRNA transcripts of the gene or genes, cDNA reversetranscribed from the mRNA, cRNA transcribed from the cDNA, DNA amplifiedfrom the genes, RNA transcribed from amplified DNA.

In some methods, a nucleic acid sample is the total mRNA isolated from abiological sample; in other instances, the nucleic acid sample is thetotal RNA from a biological sample. The term “biological sample”, asused herein, refers to a sample obtained from an organism or fromcomponents of an organism, such as cells, biological tissues and fluids.In some methods, the sample is from a human patient. Such samplesinclude sputum, blood, blood cells (e.g., white cells), tissue or fineneedle biopsy samples, urine, peritoneal fluid, and fleural fluid, orcells therefrom. Biological samples can also include sections of tissuessuch as frozen sections taken for histological purposes. Often twosamples are provided for purposes of comparison. The samples can be, forexample, from different cell or tissue types, from different individualsor from the same original sample subjected to two different treatments(e.g., drug-treated and control).

Any RNA isolation technique that does not select against the isolationof mRNA can be utilized for the purification of such RNA samples. Forexample, methods of isolation and purification of nucleic acids aredescribed in detail in WO 97/10365, WO 97/27317, Chapter 3 of LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, (P.Tijssen, ed.) Elsevier, N.Y. (1993); Chapter 3 of Laboratory Techniquesin Biochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part 1. Theory and Nucleic Acid Preparation, (P. Tijssen, ed.)Elsevier, N.Y. (1993); and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, N.Y., (1989); CurrentProtocols in Molecular Biology, (Ausubel, F. M. et al., eds.) John Wiley& Sons, Inc., New York (1987-1993). Large numbers of tissue samples canbe readily processed using techniques known in the art, including, forexample, the single-step RNA isolation process of Chomczynski, P.described in U.S. Pat. No. 4,843,155.

In gene expression analyses utilizing the devices that are described, asignificant factor affecting the results is the concentration of thenucleic acid in the sample. At low copy number, noise is related to thesquare root of copy number. Thus, the level of error that is deemedacceptable governs the copy number required. The required copy number inthe particular sample volume gives the required DNA concentration.Although not necessarily optimal, quantitation reactions can beconducted with an error level of up to 50%, but preferably is less.Assuming a 1 nanoliter volume, the DNA concentrations required toachieve a particular error level are shown in Table 3. As can be seen, 1nanoliter volumes such as used with certain of the devices havesufficient copies of gene expression products at concentrations that areworkable with microfluidic devices.

TABLE 3 Gene Expression - DNA Quantity Error (%) N (Copy No.) Volume(nL) [DNA] (10⁻¹² M) 2 2500 1 4.2 10 100 1 0.17 25 16 1 0.027 50 4 10.0066

A further calculation demonstrates that the certain of the devicesprovided herein which utilize a 1 nanoliter reaction site containsufficient DNA to achieve accurate expression results. Specifically, atypical mRNA preparation procedure yields approximately 10 ug of mRNA.It has been demonstrated that typically there are 1 to 10,000 copies ofeach mRNA per cell. Of the mRNAs that are expressed within any givencell, approximately the four most common messages comprise about 13% ofthe total mRNA levels. Thus, such highly expressed messages comprise 1.3ug of mRNA (each is 4×10⁻¹² mole or approximately 2.4×10¹² copies). Inview of the foregoing expression ranges, rare messages are expected tobe present at a level of about 2×10⁻⁸ copies. If in a standard analysisthe mRNA sample is dissolved in 10 ul, the concentration of a raremessage is approximately 2×10⁷ copies/ul; this concentration correspondsto 20,000 copies per 1 nl well (or 4×10¹¹ M).

3. Methods

Because expression analysis typically involves a quantitative analysis,detection is typically achieved using one of the quantitative real timePCR methods described above. Thus, if a TaqMan approach is utilized, thereagents that are introduced (or previously spotted) in the reactionsites can include one or all of the following: primer, labeled probe,nucleotides and polymerase. If an intercalation dye is utilized, thereagent mixture typically includes one or all of the following: primer,nucleotides, polymerase, and intercalation dye.

D. Multiplexing

The array-based devices described herein (see, e.g., FIGS. 1A, 1F, 2, 3Aand 3B and accompanying text) are inherently designed to conduct a largenumber of amplification reactions at the same time. This feature,however, can readily be further expanded upon by conducting multipleanalyses (e.g., genotyping and expression analyses) within each reactionsite.

Multiplex amplifications can even be performed within a single reactionsite by, for example, utilizing a plurality of primers, each specificfor a particular target nucleic acid of interest, during the thermalcycling process. The presence of the different amplified products can bedetected using differentially labeled probes to conduct a quantitativeRT-PCR reaction or by using differentially labeled molecular beacons(see supra). In such approaches, each differentially labeled probes isdesigned to hybridize only to a particular amplified target. Byjudicious choice of the different labels that are utilized, analyses canbe conducted in which the different labels are excited and/or detectedat different wavelengths in a single reaction. Further guidanceregarding the selection of appropriate fluorescent labels that aresuitable in such approaches include: Fluorescence Spectroscopy (Pesce etal., Eds.) Marcel Dekker, New York, (1971); White et al., FluorescenceAnalysis: A Practical Approach, Marcel Dekker, New York, (1970);Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2^(nd)ed., Academic Press, New York, (1971); Griffiths, Colour andConstitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

Multiple genotyping and expression analyses can optionally be conductedat each reaction site. If quantitative PCR methods such as TaqMan isutilized, then primers for amplifying different regions of a target DNAof interest are included within a single reaction site. Differentiallylabeled probes for each region are utilized to distinguish product thatis formed.

E. Non-Nucleic Acid Analyses

While useful for conducting a wide variety of nucleic acid analyses, thedevices can also be utilized in a number of other applications as well.As indicated earlier, the devices can be utilized to analyze essentiallyany interaction between two or more species that generates a detectablesignal or a reaction product that can reacted with a detection reagentthat generates a signal upon interaction with the reaction product.

Thus, for example, the devices can be utilized in a number of screeningapplications to identify test agents that have a particular desiredactivity. As a specific example, the devices can be utilized to screencompounds for activity as a substrate or inhibitor of one or moreenzymes. In such analyses, test compound and other necessary enzymaticassay reagents (e.g., buffer, metal ions, cofactors and substrates) areintroduced (if not previously deposited) in the reaction site. Theenzyme sample is then introduced and reaction (if the test compound is asubstrate) or inhibition of the reaction (if the test compound is aninhibitor) is detected. Such reactions or inhibition can be accomplishedby standard techniques, such as directly or indirectly monitoring theloss of substrate and/or appearance of product.

Devices with sufficiently large flow channels and reaction sites canalso be utilized to conduct cellular assays to detect interactionbetween a cell and one or more reagents. For instance, certain analysesinvolve determination of whether a particular cell type is present in asample. One example for accomplishing this is to utilize cell-specificdyes that preferentially reaction with certain cell types. Thus, suchdyes can be introduced into the reaction sites and then cells added.Staining of cells can be detected using standard microscopic techniques.As another illustration, test compounds can be screened for ability totrigger or inhibit a cellular response, such as a signal transductionpathway. In such an analysis, test compound is introduced into a siteand then the cell added. The reaction site is then checked to detectformation of the cellular response.

Further discussion of related devices and applications of such devicesis set forth in copending and commonly owned U.S. Provisionalapplication No. 60/335,292, filed Nov. 30, 2001, which is incorporatedherein by reference in its entirety for all purposes.

XI. Fabrication

A. General Aspects

As alluded to earlier, the microfluidic devices that are provided aregenerally constructed utilizing single and multilayer soft lithography(MSL) techniques and/or sacrificial-layer encapsulation methods. Thebasic MSL approach involves casting a series of elastomeric layers on amicro-machined mold, removing the layers from the mold and then fusingthe layers together. In the sacrificial-layer encapsulation approach,patterns of photoresist are deposited wherever a channel is desired.These techniques and their use in producing microfluidic devices isdiscussed in detail, for example, by Unger et al. (2000) Science288:113-116, by Chou, et al. (2000) “Integrated Elastomer FluidicLab-on-a-chip-Surface Patterning and DNA Diagnostics, in Proceedings ofthe Solid State Actuator and Sensor Workshop, Hilton Head, S. C.; and inPCT Publication WO 01/01025, each of which is incorporated herein byreference in their entirety for all purposes.

In brief, the foregoing fabrication methods initially involvefabricating mother molds for top layers (e.g., the elastomeric layerwith the control channels) and bottom layers (e.g., the elastomericlayer with the flow channels) on silicon wafers by photolithography withphotoresist (Shipley SJR 5740). Channel heights can be controlledprecisely by the spin coating rate. Photoresist channels are formed byexposing the photoresist to UV light followed by development. Heatreflow process and protection treatment is typically achieved asdescribed by M. A. Unger, H.-P. Chou, T. Throsen, A. Scherer and S. R.Quake, Science (2000) 288:113, which is incorporated herein by referencein its entirety. A mixed two-part-silicone elastomer (GE RTV 615) isthen spun into the bottom mold and poured onto the top mold,respectively. Here, too, spin coating can be utilized to control thethickness of bottom polymeric fluid layer. The partially cured top layeris peeled off from its mold after baking in the oven at 80° C. for 25minutes, aligned and assembled with the bottom layer. A 1.5-hour finalbake at 80° C. is used to bind these two layers irreversibly. Oncepeeled off from the bottom silicon mother mold, this RTV device istypically treated with HCL (0.1N, 30 min at 80° C.). This treatment actsto cleave some of the Si—O—Si bonds, thereby exposing hydroxy groupsthat make the channels more hydrophilic.

The device can then optionally be hermetically sealed to a support. Thesupport can be manufactured of essentially any material, although thesurface should be flat to ensure a good seal, as the seal formed isprimarily due to adhesive forces. Examples of suitable supports includeglass, plastics and the like.

The devices formed according to the foregoing method result in thesubstrate (e.g., glass slide) forming one wall of the flow channel.Alternatively, the device once removed from the mother mold is sealed toa thin elastomeric membrane such that the flow channel is totallyenclosed in elastomeric material. The resulting elastomeric device canthen optionally be joined to a substrate support.

B. Devices Utilizing Blind Channel Design

1. Layer Formation

Microfluidic devices based on the blind channel design in which reagentsare deposited at the reaction sites during manufacture are typicallyformed of three layers. The bottom layer is the layer upon whichreagents are deposited. The bottom layer can be formed from variouselastomeric materials as described in the references cited above on MLSmethods. Typically, the material is polydimethylsiloxane (PMDS)elastomer. Based upon the arrangement and location of the reaction sitesthat is desired for the particular device, one can determine thelocations on the bottom layer at which the appropriate reagents shouldbe spotted. Because PMDS is hydrophobic, the deposited aqueous spotshrinks to form a very small spot. The deposited reagents are depositedsuch that a covalent bond is not formed between the reagent and thesurface of the elastomer because, as described earlier, the reagents areintended to dissolve in the sample solution once it is introduced intothe reaction site.

The other two layers of the device are the layer in which the flowchannels are formed and the layer in which the control and optionallyguard channels are formed. These two layers are prepared according tothe general methods set forth earlier in this section. The resulting twolayer structure is then placed on top of the first layer onto which thereagents have been deposited. A specific example of the composition ofthe three layers is as follows (ration of component A to component B):first layer (sample layer) 30:1 (by weight); second layer (flow channellayer) 30:1; and third layer (control layer) 4:1. It is anticipated,however, that other compositions and ratios of the elastomericcomponents can be utilized as well.

During this process, the reaction sites are aligned with the depositedreagents such that the reagents are positioned within the appropriatereaction site. FIG. 6 is a set of photographs taken from the fourcorners of a device; these photographs demonstrate that the depositedreagents can be accurately aligned within the reaction sites utilizingthe foregoing approach. These photographs show guard channels andreaction site located at the end of branch flow channel. The whitecircle indicates the location of the deposited reagent relative to thereaction site. As indicated, each reagent spot is well within theconfines of the reaction site.

2. Spotting

The reagents can be deposited utilizing any of a number of commerciallyavailable reagent spotters and using a variety of established spottingtechniques. Examples of suitable spotter that can be utilized in thepreparation of the devices include pin spotters, acoustic spotters,automated micropipettors, electrophoretic pumps, ink jet printerdevices, ink drop printers, and certain osmotic pumps. Examples ofcommercially available spotters include: Cartesian Technologies MicroSys5100 (Irvine, Calif.), Hitach SPBIO (Alameda, Calif.), Genetix Q-Array(United Kingdom), Affymetrix 417 (Santa Clara, Calif.) and PackardBioscience SpotArray (Meriden, Conn.). In general, very small spots ofreagents are deposited; usually spots of less than 10 nl are deposited,in other instances less than 5 nl, 2 nl or 1 nl, and in still otherinstances, less than 0.5 nl, 0.25 nl, or 0.1 nl.

Arrays of materials may also be formed by the methods described inFoder, et al., U.S. Pat. No. 5,445,934: titled “Array ofoligonucleotides on a solid substrate”, which is herein incorporated byreference, wherein oligonucleotide probes, such as SNP probes, aresynthesized in situ using spatial light directed photolithography. Sucharrays would be used as the substrate or base of the microfluidicdevices of the present invention such that the regions of the substratecorresponding to the reaction sites, for example, blind fill chambers,would be contain one, or preferably more than one, oligonucleotideprobes arrayed in known locations on the substrate. In the case of apartitioning microfluidic structure, such as the one depicted in FIG. 15herein, the reaction sites, depicted as square boxes along theserpentine, fluid channel, would contain a plurality of different SNPprobes, preferably a collection of SNP probes suitable for identifyingan individual from a population of individuals, and preferably wherein aplurality of reaction sites along the serpentine fluid channel, suchthat if a fluid sample containing nucleic acid sequences from aplurality of individuals where introduced into the serpentine flowchannel, and a plurality of valve in communication with the serpentineflow channel such that when actuated causes the serpentine flow channelto be partitioned thereby isolating each reaction site from one anotherto contain a fraction of the fluid sample in each reaction site.Amplification of the components of the sample may be performed toincrease the number of molecules, for example nucleic acid molecules,for binding to the array of SNP probes located within each reactionsite. In some embodiments, each of the reaction sites along theserpentine fluid channel would be the same array, that is, have the sameSNP probes arrayed, and in other embodiments, two or more of thereaction sites along the serpentine t fluid channel would have adifferent set of SNP probes. Other partitioning fluid channelarchitectures could also be used, for example, branched and/or branchedbranch systems, and so forth. Other arraying techniques, such asspotting described herein, may likewise be used to form the arrayslocated within the partitionable reactions sites along a serpentine orcommon, such as in branched, fluid channel(s).

The following examples are presented to further illustrate certainaspects of the devices and methods that are disclosed herein. Theexamples are not to be considered as limiting the invention.

Example 1 Signal Strength Evaluations

I. Introduction

The purpose of this set of experiments was to demonstrate thatsuccessful PCR reactions can be conducted with a microfluidic device ofthe design set forth herein with signal strength greater than 50% of theMacro TaqMan reaction.

II. Microfluidic Device

A three layer microfluidic device, fabricated using the MSL process, wasdesigned and fabricated for conducting the experiments described in thefollowing example. FIG. 7A shows a cross-sectional view of the device.As shown, the device 700 includes a layer 722 into which is formed theflow channels. This fluid layer 722 is sandwiched between an overlayinglayer 720 that includes the control and guard layers and an underlyingsealing layer 724. The sealing layer 724 forms one side of the flowchannels. The resulting three-layer structure is affixed to a substrate726 (in this example, a slide or coverslip), which provides structuralstiffness, increases thermal conductivity, and helps to preventevaporation from the bottom of microfluidic device 700.

FIG. 7B shows a schematic view of the design of the flow channels inflow layer 722 and of the control channels and guard channel incontrol/guard layer 720. Device 700 consists of ten independent flowchannels 702, each with its own inlet 708, and branching blind channels704, each blind channel 704 having a 1 nl reaction site 706. Device 700contains a network of control lines 712, which isolate the reactionsites 706 when sufficient pressure is applied. A series of guardchannels 716 are also included to prevent liquid from evaporating out ofthe reaction sites 706; fluid is introduced via inlet 718.

II. Experimental Setup

A PCR reaction using β-actin primers and TaqMan probe to amplify exon 3of the β-actin gene from human male genomic DNA (Promega, Madison Wis.)was conducted in device 700. The TaqMan reaction consists of thefollowing components: 1× TaqMan Buffer A (50 mM KCl, 10 mM Tris-HCl,0.01M EDTA, 60 nM Passive Reference1 (PR1), pH 8.3); 3.5-4.0 mM MgCl;200 mM dATP, dCTP, dGTP, 400 mM dUTP; 300 nM β-actin forward primer andreverse primer; 200 nM FAM-labeled β-actin probe; 0.01 U/ul AmpEraseUNG(Applied Biosystems, Foster City, Calif.); 0.1-0.2 U/ul DyNAzyme(Finnzyme, Espoo, Finland); 0.5% Triton-x-100 (Sigma, St. Louis, Mo.);0.8 ug/ul Gelatin (Calbiochem, San Diego, Calif.); 5.0% Glycerol (Sigma,St. Louis, Mo.); deionized H₂O and male genomic DNA. The components ofthe reaction were added to produce a total reaction volume of 25 μl.Negative controls (Control) composed of all the TaqMan reactioncomponents, except target DNA were included in each set of PCRreactions.

Once the TaqMan reaction samples and Control were prepared, they wereinjected into microfluidic device 700 by using a gel loading pipet tipattached to a 1 ml syringe. The pipet tip was filled with the reactionsamples and then inserted into the fluid via 708. The flow channels 702were filled by manually applying backpressure to the syringe until allthe entire blind channels 704 and reaction sites 706 were filled.Control lines 712 were filled with deionized water and pressurized to15-20 psi after all of the samples were loaded into the flow lines 702,704. The pressurized control lines 712 were actuated to close the valvesand isolate the samples in the 1 nl wells 706. The guard channels 716were then filled with deionized water and pressurized to 5-7 psi.Mineral oil (15 ul) (Sigma) was placed on the flatplate of athermocycler and then the microfluidic device/coverglass 700 was placedon the thermocycler. Micro fluidic device 700 was then thermocycledusing an initial ramp and either a three-step or two-step thermocyclingprofile:

1. Initial ramp to 95° C. and maintain for 1 minute (1.0° C./s to 75°C., 0.1° C./sec to 95° C.).

2. Three step thermocycling for 40 cycles (92° C. for 30 sec., 54° C.for 30 sec., and 72° C. for 1 min) or;

3. Two step thermocycling for 40 cycles (92° C. for 30 seconds and 60°C. for 60 sec.)

MicroAmp tubes (Applied Biosystems, Foster City, Calif.) with theremaining reaction mixture, designated Macro TaqMan reactions todistinguish them from reactions performed in the microfluidic device,were placed in the GeneAmp PCR System 9700 (Applied Biosystems, FosterCity, Calif.) and thermocycled in the 9600 mode. The Macro TaqManreactions served as macroscopic controls for the reactions performed inthe micro fluidic device. The thermocycling protocol was set to matchthat of the microfluidic device, except that the initial ramp rate wasnot controlled for the Macro TaqMan reactions.

Once thermocycling was completed, the control and guard lines weredepressurized and the chip was transferred onto a glass slide (VWR, WestChester, Pa.). The chip was then placed into an Array WoRx Scanner(Applied Precision, Issaquah, Wash.) with a modified carrier. Thefluorescence intensity was measured for three differentexcitation/emission wavelengths: 475/510 nm (FAM), 510/560 nm (VIC), and580/640 nm (Passive Reference1 (PR1)). The Array Works Software was usedto image the fluorescence in the micro fluidic device and to measure thesignal and background intensities of each 1 nl well. The results werethen analyzed using a Microsoft Excel file to calculate the FAM/PR1ratio for β-actin TaqMan reactions. For conventional Macro TaqMan,positive samples for target DNA were determined using calculationsdescribed in the protocol provided by the manufacturer (TaqMan PCRReagent Kit Protocol). The signal strength was calculated by dividingthe FAM/PR1 ratio of the samples by the FAM/PR1 ratio of the controls. Asuccessful reaction was defined as a sample ratio above the 99%confidence threshold level.

III. Results

Initially, AmpliTaq Gold (Applied Biosystems, Foster City, Calif.) wasused in TaqMan reactions and FAM/PR1/Control ratios of 1.5-2.0 wereproduced, compared to Macro TaqMan reaction ratios of 5.0-14.0. Althoughresults were positive, increased signal strength was desired. Therefore,the AmpliTaq Gold polymerase was substituted with DyNAzyme polymerasedue to its increased thermostability, proofreading, and resistance toimpurities. The standard Macro TaqMan DyNAzyme concentration of 0.025U/ul was used in the microfluidic experiments. This polymerase change toDyNAzyme produced FAM/ROX/Control ratios of 3.5-5.8. The signal strengthwas improved, but it was difficult to achieve consistent results.Because it is know that some proteins stick to PDMS, the concentrationof the polymerase was increased and surface modifying additives wereincluded. Two increased concentrations of DyNAzyme were tested, 8× (0.2U/ul) and 4×(0.1 U/ul) the standard concentration for Macro TaqMan, with100 pg or 10 pg of genomic DNA per nl in the micro fluidic device.Gelatin, Glycerol, and 0.5% Triton-x-100 were added to prevent thepolymerase from attaching to the PDMS. The results of the reactions inthe micro fluidic device (chip) and the Macro TaqMan controls are shownin FIG. 8.

The microfluidic TaqMan reaction ratios range from 4.9-8.3, while theMacro TaqMan reactions range from 7.7-9.7. Therefore, the signalstrength of the TaqMan reactions in chip is up to 87% of the MacroTaqMan reactions. There was no significant difference between 4× or 8×DyNAzyme. The results demonstrate that PCR reactions can be done withgreater than 50% signal strength, when compared to the Macro TaqManreactions, in the microfluidic devices. The results have been consistentthrough at least four attempts.

Example 2 Spotting Reagents

I. Introduction

The purpose of the experiment was to demonstrate successful spotted PCRreactions in a microfluidic device. The term “spotted” in this context,refers to the placement of small droplets of reagents (spots) on asubstrate that is then assembled to become part of a microfluidicdevice. The spotted reagents are generally a subset of the reagentmixture required for performing PCR.

II. Procedure

A. Spotting of Reagents

Routine spotting of reagents was performed via a contact printingprocess. Reagents were picked up from a set of source wells on metalpins, and deposited by contacting the pins to a target substrate. Thisprinting process is further outlined in FIG. 9. As shown, reagents werepicked up from a source (e.g., microtiter plates), and then printed bybringing the loaded pin into contact with the substrate. The wash stepconsists of agitation in deionized water followed by vacuum drying. Thesystem used to print the reagent spots is a Cartesian TechnologiesMicroSys 5100 (Irvine, Calif.), employing TeleChem “ChipMaker” brandpins, although other systems can be used as described supra.

Pins employed are Telechem ChipMaker 4 pins, which incorporate anelectro-milled slot (see FIG. 9) to increase the uptake volume (andhence the number of printable spots). Under the operating conditionsemployed (typically, 75% relative humidity and temperature approximately25° C.), in excess of one hundred spots were printed per pin, perloading cycle. Under the conditions above, the volume of reagentsspotted onto the PDMS substrate is on the order of 0.1 mL.

The dimensions of the pin tip are 125×125 μm. The final spot of driedreagent is substantially smaller than this (as small as 7 μm indiameter), yet the pin size defines a lower limit to the readilyachievable spot spacing. The achievable spacing determines the smallestwell-to-well pitch in the final device. Using such a device and theforegoing methods, arrays with spacings of 180 μm have been achieved.Arrays built into working chips tend to have spacings from 600 to 1300microns.

Spotting was done using only one pin at a time. The system in use,however, has a pin head which can accommodate up to 32 pins. Printing astandard-size chip (array dimensions of order 20×25 mm) takes under 5minutes.

B. Assembly of Spotted Chips

The flow and control layers of the PCR devices are assembled accordingto the normal MSL process described above. The microfluidic devicedesign is the same as the one described in Example 1. In parallel, asubstrate layer composed of 150 μm—thick PDMS with component ratio A:Bof 30:1 is formed via spin-coating a blank silicon wafer, and then curedfor 90 minutes at 80° C.

The cured blank substrate layer of PDMS (sealing/substrate layer 724 ofFIG. 7A) serves as the target for reagent spotting. Patterns of spotsare printed onto the substrate, which is still on the blank wafer. Thereagents spotted for PCR reactions were primers and probes, specific tothe particular gene to be amplified. The spotted reagent included a1:1:1 volume ratio of 300 nM M-actin forward primer (FP), 300 mM β-actinreverse primer (RP), and 200 nM β-actin probe (Prb). In some cases, itis useful to further tune the chemistry via concentrating the spottedmixture. It has been found that adjusting the concentrations such thatprimer and probe concentrations are equal to, or slightly higher than,the normal macroscopic recipe value yields consistently good results.Therefore, the spotted reagent is concentrated to be 3 times and 4 timesthe concentration of the macro reaction. Concentration of the reagentsis performed in a Centrivap heated and evacuated centrifuge and does notalter relative FP:RP:Prb ratios. The increased spot concentrationresults in the correct final concentration when the reagents areresuspended in a 1 mL reaction volume. Spotted reagents need not belimited to primers and probes; nor must all three (FP, RP and Prb) bespotted. Applications where only the probe, or even one of the primers,is spotted can be performed. Experiments have been conducted in whichthe sample primer/probe sets spotted were TaqMan β-actin and TaqManRNAse-P.

Following the spotting process onto the substrate layer, the combinedflow and control layers (i.e., layers 720 and 722 of FIG. 7A) werealigned with the spot pattern and brought into contact. A further bakeat 80° C., for 60-90 minutes, was used to bond the substrate to the restof the chip. After the chip has been assembled, the remaining componentsof the PCR reaction (described in Example 1) are injected into the flowchannels of the chip and the chip is thermocycled as described inExample 1.

III. Results

PCR reactions have been successfully and repeatably performed usingdevices where primer (forward and reverse primers) and probe moleculesare spotted. An example of data from a chip in which a reaction has beensuccessfully performed is shown in FIG. 10.

The spotted reagents have resulted in successful PCR reactions asdefined in Example 1. Successful reactions have been performed using2-stage and 3-stage thermocycling protocols.

Example 3 Genotyping

I. Introduction

The purpose of the following experiments was to demonstrate thatgenotyping experiments can be conducted utilizing a microfluidic deviceor chip such as described herein. Specifically, these experiments weredesigned to determine if reactions conducted in the device havesufficient sensitivity and to ensure that other primer/probe sets,besides β-actin, can be performed in the microfluidic device.

II. Methods/Results

A. RNase P Experiment

RNase P TaqMan reactions (Applied Biosystems; Foster City, Calif.) wereperformed in a microfluidic device as described in Example 1 todemonstrate that other primer/probe sets produce detectable results.RNaseP reactions also require a higher level of sensitivity because theRNaseP primer/probe set detects a single copy gene (2 copies/genome) incontrast to the β-actin primer/probe set. The β-actin set detects asingle copy β-actin gene and several pseudogenes, which collectivelytotal approximately 17 copies per genome. The RNase P reactions were runwith the same components as described in Example 1, with the exceptionthat the β-actin primer/probe set was replaced with the RNase Pprimer/probe set. Further, the RnaseP primer/probe set was used at 4×the manufacturer's recommended value to enhance the fluorescence signal.The VIC dye was conjugated to the probe for RNase P and the analysisfocused on VIC/PR1 ratios. The results of one of four experiments areshown in FIG. 11.

The VIC/PR1/Control ratios for the Macro TaqMan reactions are 1.23. Thecorresponding ratios for the TaqMan reactions in the microfluidic deviceare 1.11 and 1.21. The ratios of the genomic DNA samples in themicrofluidic device are above the 99% confidence threshold level.Further, the signal strength of the TaqMan reactions in the microfluidicdevice is 50% and 93.7% of the Macro TaqMan reactions. The controlTaqMan reactions in the microfluidic device have standard deviations of0.006 and 0.012, demonstrating consistency in the reactions across themicro fluidic device. Therefore, it is determined that the TaqManreactions in the chip are sensitive enough to detect 2 copies pergenome.

B. DNA Dilution Experiment

To further determine the sensitivity of TaqMan reactions in themicrofluidic device, dilutions of genomic DNA were tested using theβ-actin primer/probe set. Reaction compositions were generally composedas described in Example 1 using 4× DyNAzyme and dilutions of genomicDNA. The genomic DNA was diluted down to 0.25 pg/nl, which correspondsto approximately 1 copy per nl. The result of one dilution series isshown in FIG. 12.

According to a Poisson distribution, 37% of the total number of wellsshould be negative if the average target number is one. Well numbers 5,6 and 7 are below the calculated threshold and, therefore, negative.This suggests that the β-actin TaqMan reactions in micro fluidic chipcan detect an average of one copy per nl. Therefore, the sensitivity ofthe reactions in the microfluidic device is sufficient to performgenotyping experiments.

C. Genotyping Experiment

Because TaqMan in the microfluidic device is capable of detecting lowtarget numbers, preliminary testing of SNP (Single NucleotidePolymorphism) genotyping was performed using the Predetermined AllelicDiscrimination kit (Applied Biosystems; Foster City, Calif.) against theCYP2D6 P450 cytochrome gene. The kit contains one primer set and twoprobes; FAM labeled for the wildtype or reference allele, CYP2D6*1, andVIC labeled for the CYP2D6*3 mutant or variant allele. Positivecontrols, PCR products, for each allele along with genomic DNA weretested in the device using the same conditions as described inExample 1. The results from one experiment are shown in FIGS. 13 and 14.The experiment has been repeated at least three times to validate theresults and to demonstrate reliability.

As shown in FIG. 13, the Al-1 (Allele 1, CYP2D6*1 wild type allele) andgenomic DNA (100 pg/nl) produced an average VIC/PR1/Control ratio of 3.5and 2.2, respectively, indicating that the genomic DNA was positive forthe CYP2D6*1, wild type allele. These values are above the thresholdlimit for the reactions. The signal strength of the TaqMan reactions inthe microfluidic device is 59% and 40% of the Macro TaqMan controls,respectively. Al-2 (Allele 2, CYP2D6*3 mutant or variant allele), whichshould be negative in the VIC channel, showed some signal over control(1.5), possibly due to FAM fluorescence leaking into the VIC channel ofthe detector. The leakage can be minimized with an improved detectionprocess.

The Al-2 positive control gave an average FAM/PR1/Control ratio of 3.0,which was 37% of the Macro TaqMan signal and above the calculatedthreshold limit (see FIG. 14). The genomic samples were negative for theCYP2D6*3 mutant allele, an expected result since the frequency of theCYP2D6*3 allele is low. Again, it appears that there is some leakage ofthe Al-1, VIC probe into the FAM channel of the detector. Overall, theSNP detection reactions were successful in the microfluidic device.

Example 4 Verification of PCR by Gel Electrophoresis

I. Introduction

As an alternative method to prove amplification of DNA was occurring inthe microfluidic device, an experiment to detect PCR product by gelelectrophoresis was performed. PCR reactions compositions were asdescribed in Example 1, except the TaqMan probe was omitted and theβ-actin forward primer was conjugated to FAM.

II. Procedure

A. Microfluidic Device

A three layer microfluidic device, fabricated using the MSL process, wasdesigned and fabricated for conducting the experiments described in thisexample; FIG. 15 shows a schematic view of the design. The device 1500generally consists of a sample region 1502 and a control region 1504.Sample region 1502 contains three hundred and forty-one 1 nl reactionsites 1508 represented by the rectangles arrayed along flow channel1506, which includes inlet via 1510 and outlet via 1512. Control region1504 contains three control flow channels 1514 each containing ten 1 nlreaction sites 1518, also represented by the rectangles and an inlet via1516. A network of control lines 1522 isolate each reaction site 1508,1518 when sufficient pressure is applied to inlet via 1524. A series ofguard channels 1520 are included to prevent liquid from evaporating outof the reaction sites 1508, 1518. The device is a three-layer device asdescribed in Example 1 (see FIG. 7A). The entire chip is placed onto acoverslip.

B. Experimental Setup

Microfluidic device 1500 was loaded and thermocycled using the 3temperature profile described in Example 1. The remaining reactionsample was thermocycled in the GeneAmp 9700 with the same thermocyclingprofile as for microfluidic device 1500. The reaction products wererecovered after thermocycling was completed. To recover the amplifiedDNA, 3 μl of water was injected into sample input via 1506 and 3-4 μl ofproduct were removed from outlet via 1512. The reaction products fromdevice 1500 and the Macro reaction were treated with 2 μl of ExoSAP-IT(USB, Cleveland, Ohio), which is composed of DNA Exonuclease I andShrimp Alkaline Phosphatase, to remove excess nucleotides and primers.The Macro product was diluted from 1:10 to 1:106. The product fromdevice 1500 was dehydrated and resuspended in 4 μl of formamide.

III. Results

Both products, along with negative controls were analyzed, on apolyacrylamide gel. FIG. 15 shows the gel electrophoresis results. Theappropriate size DNA band of 294 base pairs in length is observed inFIG. 16.

The products from the Macro reactions are shown on the left hand side ofthe gel and correspond to about 294 base pairs, the expected size of theβ-actin PCR product. The negative controls lack the PCR product.Similarly, the product derived from the device gave the expected β-actinPCR product. Therefore, target DNA was amplified in the micro fluidicdevice.

Example 5 Massive Partitioning

The polymerase chain reaction (PCR) has become an essential tool inmolecular biology. Its combination of sensitivity (amplification ofsingle molecules of DNA), specificity (distinguishing single basemismatches) and dynamic range (10⁵ with realtime instrumentation) makeit one of the most powerful analytical tools in existence. Wedemonstrate here that PCR performance improves as the reaction volume isreduced: we have performed 21,000 simultaneous PCR reactions in a singlemicrofluidic chip, in a volume of 90 μL per reaction and with singletemplate molecule sensitivity.

FIGS. 17 a-17 d depict a single bank and dual bank partitioningmicrofluidic device. where multilayer soft lithography (SL) (1), wasused to create elastomeric microfluidic chips which use active valves tomassively partition each of several liquid samples into a multitude ofisolated reaction volumes. After injection of the samples into inlet1703 which is in communication with branched partitioning channel system1705 of microfluidic device 1701 (FIG. 17 b), 2400 90 pL volumes 1709 ofeach sample are isolated by closing valves 1707 spaced along (FIG. 17 d)simple microfluidic channels. The chip device is then thermocycled on aflat plate thermocycler and imaged in a commercially availablefluorescence reader.

We assessed the performance of PCR in the chips by varying theconcentration of template DNA and measuring the number of wells thatgave a positive Taqmant™ signal. We found that a digital amplificationis observed when the average number of copies per well is low (FIGS. 18a and 18 b). A mixture of robust positive and clearly negative signalsis observed even when the average number of copies per well is below 1;this implies that even a single copy of target can give goodamplification. The number of positive wells was consistent with thenumber of wells calculated to have ≧1 copy of target by the Poissondistribution (FIG. 19). This result validates that this system givesamplification consistently even from a single copy of target.Fluorescent signal strengths from microfluidic Taqman™ PCR werecomparable to macroscopic PCR reactions with the same DNAconcentration—even though the macroscopic reactions contained >10⁴ moretemplate copies per reaction.

We believe that the primary source of this remarkable fidelity is theeffective concentration of the target: a single molecule in a 90 pLvolume is 55,000 times more concentrated than a single molecule in a 5uL volume. Since the number of molecules of target, n_(t), does notchange (i.e. n_(t)=1) and the number of molecules that can produce sidereactions, n_(s), (i.e. primer-dimers and non-complementary DNAsequences in the sample) is linearly proportional to volume (i.e.n_(s)∝V), the ratio of target to side reactions is inverselyproportional to volume: n_(t)/n_(s)∝1/V. Since side reactions are aprimary cause of PCR failure (4), the advantage to reducing the volumeof the reaction is clear.

PCR amplification from single copies of template has been previouslyreported (5). However, current methods that achieve reliableamplification from single copies in a macroscopic volumes often requirealtered thermocycling protocols (e.g. long extension times, manycycles), precautions against mispriming and non-specific amplification(e.g. “hot start” PCR (thermal activation of the polymerase), “booster”PCR, additives to reduce nonspecific hybridization, etc), and are almostalways done with two rounds of PCR, where an aliquot of the first PCR isused as template in the second reaction. In contrast, this systemachieves reliable amplification from single copies using standardconditions—off-the-shelf primers and probes and a single-round, standardthermocycling protocol. Being completely enclosed, it is also nearlyinvulnerable to environmental contamination. The ability to do massivenumbers of PCR reactions simultaneously provides definite logistical,cost and time advantages compared to macroscopic volumes (1 chip with21,000 reactions vs. 219 separate 96 well plates, and the associatedtime, equipment, and tracking infrastructure).

This principle of massive partitioning with a digital PCR readout may beused for absolute quantification of the concentration of target in asample. It can be used, for example, to genotype a pooled sample ofgenomic DNA simply by counting the numbers of wells that give a positivefor a particular allele, or plurality of alleles as described above. Dueto the enhanced resistance to side reactions, it should also be usefulin quantifying mutants in a background of wild-type DNA—a problemrelevant in cancer detection. The general principle of concentration bypartitioning may also be useful in other reactions where detection ofsingle molecules, bacteria, viruses or cells is of interest (e.g. ELISAreactions for protein detection). Digital PCR is described by Brown, etal., U.S. Pat. No. 6,143,496, titled “Method of sampling, amplifying andquantifying segment of nucleic acid, polymerase chain reaction assemblyhaving nanoliter-sized chambers and methods of filling chambers”, and byVogelstein, et al, U.S. Pat. No. 6,446,706, titled “Digital PCR”, bothof which are hereby incorporated by reference in their entirety. Thesmall volumes achievable using microfluidics allow both a massive degreeof parallelization and very high target-to-background concentrationratios. High target-to-background ratios allow single-moleculeamplification fidelity. These factors suggest that for PCR, smallerreally is better.

The invention provides for methods and devices for conducting digitalPCR in a microfluidic environment comprising the steps of: providing amicrofluidic device having a fluid channel therein, said fluid channelhaving two or more valves associated therewith, the valves, whenactuated, being capable of partitioning the fluid channel into two ormore reaction sites or chambers; introducing a sample containing atleast one target nucleic acid polymer, actuating the valves to partitionthe fluid sample into two or more portions, wherein at least one portioncontains a target nucleic acid polymer and another portion does notcontain a target nucleic acid polymer, amplifying the target nucleicacid polymer, and, determining the number of portions of the fluidchannel that contained the target molecule. In preferred embodiments,the microfluidic device comprises an elastomeric material, and morepreferably, comprises at least one layer comprising an elastomericmaterial. In certain preferred embodiments, the microfluidic devicefurther comprises a deflectable membrane wherein the deflectablemembrane is deflectable into and out of the fluid channel to controlfluid flow within the fluid channel and/or to partition one portion ofthe fluid channel from another, preferably wherein the deflectablemembrane is integral to a layer of the microfluidic device having achannel or recess formed therein, and preferably wherein the deflectablemembrane is formed where a first channel in a first layer is overlappedby a second channel in a second layer of the microfluidic device. Insome embodiments, the sample fluid contains all of the components neededfor conducting an amplification reaction, while in other embodiments,the microfluidic device contains at least one component of anamplification reaction prior to the introduction of the sample fluid. Insome embodiments, the microfluidic device further comprises a detectionreagent, preferably one or more nucleic acid polymers complimentary to aleast a portion of the target nucleic acid polymer, preferably aplurality of different nucleic acid polymers spatially arrayed within areaction site or chamber of the microfluidic device.

Amplification may be achieved by thermocycling reactions such as PCR, orby isothermal reactions, such as described by Van Ness et al., in U.S.patent application Ser. No. 10/196,740 which has publishes as US2003/0138800 A1, which is herein incorporated by reference in itsentirety for the purpose of teaching an isothermic amplification scheme.

The invention further provides for a protein microcalorimetry assayusing a fluorescent dye, for example SYBER Green™, to measure theconformational changes of a protein, such as denaturation, especially ifa protein's denaturation temperature changes when the protein interactswith another moiety such as a ligand or compound or other protein. Anadditional benefit of using SYBR Green™ is that it us used at lowerwavelengths than other UV range dyes thus reducing background problemstypically associated with many plastic materials.

Example 6 K-Ras: Detection of Mutant Target in High Background

K-ras codon 12 mutant genomic DNA was obtained from MIA PaCa2 cell linewas obtained from the Allan Balmain Laboratory at the University ofCalifornia, San Francisco. The mutant genomic DNA was extracted usingstandard extraction protocols. For controls, one cell line, HELA (ATCC)was cultured and its DNA extracted per standard protocol, and a secondcontrol was obtained as purified human genomic DNA from PROMEGA™.

Reaction cocktails contained 50% of 2× master mix from a TAQMAN™ kitsold by ABI™ corporation, a final concentration of 0.25% TWEEN™ fromSIGMA™ corporation, mutation specific primers (e.g., 3′-TC CC GCA TCCGTT CTC ACG-5′) at a concentration of 800 mM, wild-type non-extendibleprimer at 500 nM, a universal probe at 200 nM designed to function inaccordance with the TAQMAN™ protocol specific for the presentapplication, the balance being made up with DI water. The cocktail wasdivided into several different aliquots, which each received an amountof target genomic DNA and/or “background” DNA. Specifically, the firstreaction (“Aliquot 1”) was a no-template control which received no DNAto establish a baseline signal. The second aliquot (“Aliquot 2”)received 100 pg of mutant target DNA to serve as a positive control. Thethird aliquot (“Aliquot 3”) received 100 pg of mutant DNA along with 500ng of wild-type (from PROMEGA™) DNA. The forth aliquot (“Aliquot 4”)received 100 pg of mutant DNA along with 1 μg of HELA (ATCC) wildtypeDNA. The fifth aliquot (“Aliquot 5”) received 100 pg of mutant DNA alongwith 1 μg of wild type (PROMEGA™) DNA. The fourth and fifth aliquotswere to demonstrate the ability of the system to discern lowconcentrations of target among two different types of relatively highconcentration “background” DNA. And, the sixth aliquot (“Aliquot 6”)received 1 μg of wild type (PROMEGA™) DNA only as negative control.

The chip used had twelve sample channels, each channel having 1,200partitioning valves to create approximately 1,200 isolated chambers fromeach sample loaded into each sample channel through blind filling. Onceeach sample had been loaded into its respective sample channel, thesample channels were partitioned by applying a hydraulic force to thecontrol channels that formed the partitioning valves along each samplechannel. In this particular instance, the hydraulic fluid comprisedwater and PEG 3,350 mw average (from HAMPTON RESEARCH™ corporation) at aconcentration of 25% w/v. The hydraulic fluidic was pressurized to apressure of 35 PSI and held closed during the PCR reaction.

Amplification by PCR was performed in real-time using a modifiedthermocycler that had a suction metallic block having a tortuous channelsystem thereon, the metallic block being disposed between the side ofthe silicon wafer opposite the elastomeric block and the thermal controlsurface of the thermocycler, wherein a vacuum source was applied to thetortuous channel system to urged the silicon wafer into near homogeneousthermal contact between the silicon wafer and the thermocycler thermalcontrol surface as described in co-pending patent application Ser. No.11/043,895, filed on Feb. 14, 2005, which is herein incorporated byreference in its entirety. As an alternative to using the vacuum chuckto hold down the chip and to establish thermal contact, a film of oil,such as mineral oil, may be used to achieve thermal conduction betweenthe thermal control surface and the exposed silicon portion of the chip.

A thermocylcling profile comprising 2 minute UNG clean up step at 52degrees C., followed by a “hot start” at 95 degrees C. for ten minutes,followed by forty cycles of ramping between 25 seconds at 95 degrees C.to 40 seconds at 58 degrees C., operating the thermocycler at itsfastest ramp rate between each temperature change.

At the end of each 58 degree C. round, two different images were takenof the chip using white light source that was filtered in accordancewith the two different fluorophores used for the two color analysis.FIG. 22 shows fluorescent images of portions of each of six channels ina DID chip (approximately one-half of the 1,200 isolated chambers ineach channel) in which K-ras codon 12 mutant-specific amplification wascarried out.

Ratio analysis was conducted between the two different color images toproduce real-time amplification curves. FIG. 23 shows that mutantamplification products were detected after 30 PCR cycles. FIG. 24 showsthat additional amplification products (false positives) were detectedafter about 35 PCR cycles.

Another experiment was performed using a chip similar to theabove-mentioned chip, however, this chip one sample channel able topartition the sample channel into approximately 10,000 separatechambers. The sample comprises a cocktail as described above, andfurther including 100 pg of mutant DNA and 7 μg of wild type HELA DNA.The number of cycles of amplification in this instance was only 30, ofwhich the last cycle included an extension period of five minutes at 72degrees C. Fluorescence from amplification of mutant alleles wasdetected in individual chambers. Although the experiment was monitoredin real-time as described above, it was appreciated that the assay couldbe performed as an end-point type assay.

Example 7 BCR-Abl: Detection of Mutant Target in High Wild-TypeBackground

A similar experiment to that described above was performed to detect lowlevels of an Abl tyrosine kinase mutant in a very high background ofwild-type (WT) Abl cDNA. Point mutations in the Abl tyrosine kinasedomain are the main mechanism of secondary resistance to ImatinibMesylate (IM) therapy in chronic myeloid leukemia (CML) patients.Clinically, the experimental goals were to identify mutations inpre-treatment and very early treatment samples using the DID chip withsuperior sensitivity and specificity.

The chip used was a PDMS microfluidic chip mounted on a moldedSBS-standard-plate formatted carrier with inputs and pressureaccumulator to act as an interface between the user and the PDMS chip. Aheat exchange plate (“Integrated Heat Spreader”) is placed underneathand in contact with the chip to ensure rapid heat transfer andtemperature uniformity within the array. In the present example thereare 12 carrier inputs corresponding to 12 separate sample inputs to thechip. Individual samples (minimum 6.6 μl volume each) are delivered intopre-programmed locations in the chip by pressure-driven “blind filling”in the PDMS. Each sample is distributed into 1104 independent 6 nlwells. Control lines are primed with control fluid and are pressurizedto actuate the valves between the wells. The valves partition theindividual wells and are kept closed during the PCR experiment.

The microfluidic chip was used to partition a sample into hundreds,thousands, or tens of thousands of isolated reaction chambers, therebyenriching rare target mutant polynucleotides relative to wild-typepolynucleotides and enabling absolute quantitative, specific andsensitive detection of rare sequences and mutations.

Mutation-specific PCR assays were developed for the following mutations:G250E, Q252H (2 substitutions), Y253F, E255K, E255V and T315I. Initialexperiments utilized mutated BCR-Abl cDNA and plasmids serially dilutedinto a background of WT BCR-Abl cDNA (from a transformed BaF3 cellline). The ration of mutant to wile-type was about 1:1×10⁵. FIG. 25shows the BCR-Abl tyrosine kinase domain mutations and exploded view ofexons 4 and 6, with primers and probes used in PCR assays indicated.

By performing massive partitioning and PCR on a single microfluidicchip, single copies of the mutant gene were specifically and reliablydetected in each of the mutation-specific assays in a background of 10⁵WT copies. Total Abl polynucleotide was also quantified to allow for thedetermination of percent mutant Abl in the assays applied to clinicalsamples.

Results are shown in FIGS. 26 to 28. FIG. 26 shows a fluorescent imageof the DID chip after 32 cycles of mutation-specific PCR assay for Q252HG>C and Q252H G>T. FIGS. 27A and 27B shows images of DID chips afterindicated cycles of mutation-specific PCR assays for T315I and Y253F.Single mutant cDNA copies were detected in background of up to 10⁵ WTBCR-Abl cDNA copies. A) T315I-specific TaqMan reactions containedmutant-specific primers, Black Hole Quencher® probe with 6-FAM reporter.B) Y253F-specific TaqMan reactions contained mutant-specific primers,Black Hole Quencher Probe® with Cal-Fluor Orange® reporter.

FIGS. 28A and 28B shows fluorescent images of DID chip after 39 cyclesof T315I-Specific PCR and Total BCR-Abl-Specific PCR assays. A) T315IMutant-specific TaqMan PCR on DID chip: single mutant copies detectedand quantified as indicated in complex background of greater than 10³BCR-Abl copies. B) Total BCR-Abl amplified with primers exterior tocodon 315 by TaqMan PCR on DID chip: Saturated signal points out greaterthan 10³ copies.

BCR-Abl cDNA for wild-type (WT) (T315I and Y253F) was prepared from BaF3cells by BCR-Abl-specific reverse transcription reaction. BCR-Abl cDNAfor wild-type (WT) (T315I and Y253F) was prepared from BaF3 cells byBCR-Abl-specific reverse transcription reaction. BCR-Abl plasmid DNAwith mutations G250E, Q252H G>C, Q252H G>T, E255K, and E255V werediluted by WT cDNA to represent clinical model mixtures. Totalrandom-primed cDNA was prepared from CML patient blood leukocytes.TaqMan 5′ nuclease assays to specifically amplify the p-loop mutationsand the T315I mutation comprised of the following: Mutation-specificprimers with 2 mismatches relative to WT and 1 mismatch relative toMutant WT-specific non-extendable primer for codon 315 (3 terminaldideoxy-C) with 2 mismatches relative to Mutant and 1 mismatch relativeto WT One Black Hole Quencher probe specific to exon 4 (p-loop) andanother specific to exon 6 (T315I).

The present experiment demonstrates that an elastomeric microfluidicchip can be used to perform massive partitioning followed by PCR toprovide highly sensitive and specific detection of known pointmutations. Single mutant copies can be specifically and reliablydetected in mutation-specific assays in mixtures in which the ratio ormutant to wild-type is about 1:1×10⁵. This sensitivity is at least 50times more sensitive than current conventional methods. This inventionis a very significant improvement over the pervious technology andprovides a promising method for early detection of low-level mutant geneexpression.

In the specific case of the Abl tyrosine kinase mutant, the presentinvention may allow patient therapy to be altered before resistance isdetected cytogenetically and before disease progression is evident.Earlier treatment may lead to more effective therapy and greatersurvival.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

The invention claimed is:
 1. A method for highly sensitive and specificdetection of mutant alleles in a high background of wild-type alleles,comprising a) obtaining an assay composition comprising firstpolynucleotides encoding a mutant allele of a gene and secondpolynucleotides encoding a wild-type allele of the gene, wherein theratio of first polynucleotides to second polynucleotides is 1:1×10³ orlower; b) distributing the assay composition, or a portion thereof, intoat least 10,000 aliquots, thereby producing a subset of aliquots inwhich the ratio of first polynucleotides to the second polynucleotidesis greater than the ratio of first polynucleotides to secondpolynucleotides in said assay composition, wherein some aliquotscomprise a first polynucleotide and essentially all aliquots comprise atleast one second polynucleotide; c) combining the assay composition withamplification reagents prior to step (b) or combining the at least10,000 aliquots with amplification reagents after step (b), wherein saidamplification reagents: i) comprise amplification primers specific foramplifying at least a portion of the mutant allele, and ii) do notcomprise amplification primers specific for amplifying the wild-typeallele; d) amplifying the at least a portion of the mutant alleleencoded in the first polynucleotides; and e) detecting the presence orabsence of amplification in each aliquot.
 2. The method of claim 1wherein the first and second polynucleotides are derived from the genomeof the same individual.
 3. The method of claim 1 wherein said assaycomposition is combined with all of said amplification reagents prior tostep (b).
 4. The method of claim 1 wherein the mutant allele differsfrom the wild-type allele by one or more point mutations.
 5. The methodof claim 1 wherein the mutant allele differs from the wild-type alleleby an insertion or deletion.
 6. The method of claim 1 wherein theamplification in (d) is by real-time PCR.
 7. The method of claim 1wherein the amplification mixture additionally comprises a nonextendibleprimer that binds specifically to the wild-type polynucleotide.
 8. Themethod of claim 1 wherein the ratio of first polynucleotides to secondpolynucleotides is 1:1×10⁵ or lower.
 9. The method of claim 1, whereinthe presence of amplification is determined by detecting a fluorescentsignal.
 10. The method of claim 1 wherein the gene is an oncogene. 11.The method of claim 1 wherein the assay composition is combined withamplification reagents prior to step (b).
 12. The method of claim 1wherein some aliquots comprise a first polynucleotide and essentiallyall aliquots comprise at least a plurality of second polynucleotides.13. The method of claim 12 wherein some aliquots comprise a firstpolynucleotide and essentially all aliquots comprise hundreds of secondpolynucleotides.